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Rihanna "Ignoring" Untrue Hassan Jameel Split Reports
Rihanna is "ignoring" all the untrue Hassan Jameel split stories, Gossip Cop has exclusively learned. As we were first to report, despite claims by lesser outlets, Rihanna has not dumped her boyfriend. The fake news was first spread by a site that over the years has falsely maintained, for example, that Rihanna was pregnant with Prince Harry and Leonardo DiCaprio's babies.
On Friday, MediaTakeOut asserted Rihanna was "back on the market" because she "dumped" her super wealthy boyfriend. The often discredited outlet alleged the Barbadian singer and Ocean's 8 star simply "got tired" of Jameel. Glaringly, the website had no details about when, how, or where the supposed breakup took place. Did she do it on Friday, the week before, or a month ago? Did Rihanna dump Jameel over the phone, by text, or in person? And if done by person, was it done in Los Angeles, Saudi Arabia or France?
The website had no answers to any of those Journalism 101 questions. And yet places like HollywoodLife and even respected magazines, including Elle and Harper's Bazaar, readily picked up the report without bothering to fact-check. Nowhere in any of their articles is it mentioned that the alleged split was confirmed by Rihanna's rep or any particular insider. Conversely, a mutual acquaintance of the singer and Gossip Cop assured us on background that talk of Rihanna having split with Jameel was "untrue."
Even though the false rumor was picked up and republished by several outlets, Gossip Cop's impeccable insider says Rihanna is "ignoring" the inaccurate stories. Nor is she going out of her way to debunk the inaccurate articles. We're told Rihanna prefers to keep her relationship with Jameel private, but eventually it will be obvious the reports are wrong. To date, with a few exceptions such as when Rihanna and Jameel were hanging out on a balcony in Paris, the two have rarely been photographed together.
And perhaps Rihanna is right to ignore the noise because MediaFakeOut's initial report has spawned a number of wholly fabricated tales. In fact, HollywoodLife exposes its questionable practices with a new story titled, "Rihanna: Would She Take Chris Brown Back If She's Really Split From Hassan? The Truth, Says Source." While clearly trying to find a new angle, the site alleges to know whether Rihanna would take back Chris Brown, who plead guilty in 2009 to assaulting her.
The often discredited outlet, which is nicknamed HollywoodLies because of its penchant for fabricating, acknowledges that it's unsure "if [Rihanna's] really split from Hassan." It further references "reports of a breakup" and notes it doesn't know for certain "if she is indeed a single lady once again." Yet, after admitting it's unsure whether Rihanna actually split with Jameel, it claims to have a "Rihanna" source. Of course, if HollywoodLies truly had a Rihanna "source" it would know for sure whether the singer and Jameel split instead of saying "reports of a breakup." Perhaps Rihanna's tact is right, and it's best to ignore all the fake news written about her.
Lewittes, Michael. "Rihanna, Boyfriend Hassan Jameel Have NOT Split, Despite Claims She 'Dumped' Him." Gossip Cop, 1 June 2018.
Heller, Corinne. "Rihanna Turns 30: Inside Her Private Romance With Hassan Jameel." E! Online, 20 Feb. 2018.
"Rihanna dons Burberry hat while she and boyfriend Hassan Jameel enjoy romantic getaway to Paris." Daily Mail.


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Coconut Chocolate Chip Cookies
I found the recipe for these cookies on the bag of a Chocolate Chip bag, but of course I had to put my stamp on them, and so I made some healthy adjustments.
I always store my cookies in the freezer, as to ensure there are always cookies on hand. A sure fire way to make sure you do not eat the entire batch of cookies it to keep them out of your minds eye; out of sight out of mind. Having cookies on hand serves another purpose other than binging yourself into a diabetic coma, you never know when someone will come for tea, and so I like to be ready. Also these freezer cookies make a great addition to my hot morning coffee.
Now I wouldn't recommend you go ahead and eat a stack of cookies in one sitting, BUT if you are going to indulge (and we all do) these cookies are not so bad. I've made some healthy substitutions by skipping the butter and using apple sauce (zero saturated fat); I also downgraded the brown sugar by 1/4 cup, and the white sugar by 1/2. Unsweetened Coconut is also a great way to add flavour and texture without extensive added sugar or fat. You could also use raisins instead of the semi-sweet chips, but that entirely up to you.
| Recipe |
1 cup of apple sauce
3/4 cup of brown sugar
1/2 cup of white sugar (Splenda also works here)
2 eggs
1 1/2 tsp of vanilla extract
2 cups flour
1 tsp baking soda
1/2 tsp salt
1 bag of semi-sweet chocolate chips
1/2 cup of unsweetened coconut
Beat together apple sauce and sugars, then mix in eggs, and vanilla. Combine dry ingredients and slowly add to wet mixture. Add chocolate chips, and coconut.
Drop combined ingredients by the tablespoon onto baking sheet lined with parchment paper, and bake on 375 for 10 minutes.
Source: http://feedproxy.google.com/~r/ShortPresents/~3/tcP97DkFtx0/bag-cookies.html

subset: commoncrawl


text: The newest in I Love Mel's pop series, featuring lots of activities and colouring in to cosy up to the lovely Antoni (figuratively, of course). Antoni Porowski is a handsome avocado and cocktail expert who is witty, charming, sensitive and emotional at times - and buff as hell! What better way to celebrate 'Antoni the Great' than to colour him good? This interactive activity book includes lots of images of Antoni to be coloured in as well as facts, fun and games. For all fans of all things Queer Eye.


text: I have a VW with leather seats. The centers are leatherette. The color is light beige. I decided to spray Kiwi Camp Dry silicone on the centers. The overspray got on the smooth beige leather. By the next day, the smooth leather yellowed. I have tried cleaning it with Lexol leather cleaner. It did help but not near enough. Thought I better get advise before trying something else.


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By Michael S. Carl, Andini Dewi and Bambang Dhanisworo Defamation under Indonesian law involves the issuance of a defamatory statement that causes a person to suffer harm. The term defamation is often used to encompass both slander (penistaan) and libel (penistaan dengan surat). Defamation is governed in the Indonesian Criminal Code under Chapter XVI, which covers six types of defamation. Defamation under the Indonesian Criminal Code 1. Slander or defamation (Article 310 (1) of the Criminal Code). 2. Libel or defamation in writing (Article 310 (2) of the Criminal Code). The elements that need to be fulfilled under Article 310 (1) and (2) are:
- the defamation is made intentionally to harm a person's honor or reputation
- the defamation is addressed to an individual, not a government institution, official or association, community, etc.
- the defamation needs to contain an accusation about a specific matter (e.g., deception, embezzlement, etc.), as long as it disgraces the related person and
- the defamation is made publicly.


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What We Do
All of our solar traffic products adopt mechanization production to improve t he quality, Most overseas customers feedback our products are good quality
Hot Products
Street Name Plates and Numbers. The Council is responsible for ensuring that street name plates are provided and fitted in suitable positions.
who we are
Hunan Xiangxu Traffic & Lighting Hi-Tech Co.,Ltd is a professional company which are specialized in all kinds of road safety equipment, such as Traffic Signs, Solar LED Traffic Lights,Solar LED Traffic Signs,Road Studs,Reflectors, Road Barriers, Traffic Cones, ect.&D team.more


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I have been away for several weeks due to some troubling news that my wife and I received regarding her pregnancy. She is doing well but there is a good chance that she is in the early stage of Placenta Accreta. This will present a particular set of difficulties down the road as she gets closer to her due date. In light of this, my time has been occupied with taking care of her, our home, our children. It has also presented the opportunity to reflect on God's providence and unceasing love for those who are called to be His.
I confess that I struggled at first to understand why this was happening. I would imagine this is a fairly normal reaction for most people, Christian and non-Christian alike. In a single instance everything seemed to come to a screeching stop. Things that seemed important before now appeared trivial and insignificant. And the nagging question continued to haunt me: "Why would God, my Father in Heaven, allow us to go through such an ordeal?" As I was driving home from work one afternoon I was flooded with a terrible anxiety and my imagination ran wild… But only for a few moments.
At this point, my theology kicked in. This is what this blog is about: where the study and the "everyday" meet. I immediately and intentionally began to meditate on some biblical and theological truths that served as a salve for my soul. I remembered that I love God, and that He causes all thing to work out for our good (Rom. 8:28). I reflected on God's sovereign grace and a love that neither my wife or I could ever be separated from (Rom. 8:38-39). I thought about the role that trial and tribulations play in our lives and how God uses these to build us up in our most holy faith; how they should not rob of us joy (James 1:2-3; Rom. 5:3). All of these are founded on theological truths about the nature and being of God, His providential hand in all things, His lordship over all creation, but most of all, in the blessed gospel of God's saving grace in Christ. The truth is that I cannot fathom how the unbeliever can graciously face the difficulties and anxieties that accompany this life without gospel of Jesus, without a biblical theology of suffering. Regardless of the outcome, Christ is mine and I am His. Adding to that the fact that my wife is also a believer brings all the more joy.
Suffering, trials, tribulations, afflictions…these are God's ordained means of growth in the life of the Christian (Acts 14:22; 1 Peter 4:12; 2 Tim. 3:12). No other religion in the world would make such a claim. It forces us to face our own sinfulness and fall into the hands of the Creator, Sustainer, and Redeemer of our bodies and souls. Does this mean we do not feel the pain of suffering? No, in this life there will be pain and heartache. Concern for our loved ones will always be there. But underneath it all, there is an unshakable joy in knowing that the God of the Scriptures has us in His heart and mind, and He will use these disconcerting moments to increase our faith, our holiness, and make us more like the Savior. Therefore, we can be strong and courageous in the Lord (Deut. 31:6; Eph. 6:10).
Blessed be the God and Father of our Lord Jesus Christ, the Father of mercies and God of all comfort, who comforts us in all our affliction, so that we may be able to comfort those who are in any affliction, with the comfort with which we ourselves are comforted by God. For as we share abundantly in Christ's sufferings, so through Christ we share abundantly in comfort too.
2 Corinthians 1:3-5


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Heavy-duty drivers, meet the 2019 Ford F-350. If you plan to tow and haul the kinds of loads that a lesser truck can't handle, you've come to the right place. This heavy-duty truck is specially designed to suit heavy-duty drivers.
Depending on configuration, you can load the bed with up to 7,630 pounds of timber, rocks, machinery, and whatever else needs hauling. The F-350's best-in-class tow ratings mean you can move gooseneck trailers weighing up to 34,000 pounds. Inside, the recently updated cabin - whether regular, extended, or crew - provides spacious, supportive seating and every imaginable creature comfort. This heavy-duty truck is also known for its unique driver assist features. For example, the cruise control system can maintain a smooth, comfortable ride, even with 30,000 pounds in tow. You can program the lane-keeping assist and blind-spot monitoring features to take the length of your trailer into consideration, thereby creating a safer ride overall.
The F-350 is a heavy-duty workhorse, it's true, but it's also a plush family hauler. Choose from six trim models - the XL, XLT, Lariat, King Ranch, Platinum, and Limited - for the creature comforts you crave. While the base XL offers the basis of a good work truck, the XLT adds modern creature comforts, like cloth seating and keyless entry. The Lariat and up are classier, providing upscale extras like leather upholstery and standard infotainment technology and smartphone integration.
In addition to trim models, you also must select the cabin and bed size combo that works for your daily ride. Regular, extended, and crew cabins offer wide, supportive seats. The extended and crew cabs provide backseat seating, so you can transport your crew from job site to job site. Choose between the 6.8-foot and 8.2-foot beds to haul your work supplies.
This Super Duty truck tows best-in-class loads. Depending on configuration, you can hook up to 21,000 pounds up to the conventional tow rig. Tow your four-horse trailer or massive toy-hauler and travel across country with ease. Equipped for fifth-wheel towing, your 2019 Ford F-350 can tow up to 27,500 pounds. That's the kind of utility you need to move heavy machinery, like farming machinery or a trailer full of raw timber. Add the gooseneck hitch prep package, and this heavy-duty truck can tow gooseneck trailers weighing up to 34,000 pounds, a best-in-class figure.
Again, the answer to this question depends on configuration. Max payloads range from 4,470 to 7,630 pounds. When you're choosing between cab sizes and trim models, make sure you select a combination that's capable of hauling your company supplies and machinery.
One of the biggest, boldest trucks on the market, the 2018 Ford F-350 is now lighter and more maneuverable.
When you pit the 2018 Ford F-350 against the Ram 3500, which heavy-duty truck comes out on top? The answer to this question might surprise you. Before you start shopping for a heavy-duty truck that tows and hauls gargantuan loads, give our comparison guide a spin.
When you've got massive weight to move, look to the 2019 Ford F-350 and Chevrolet Silverado 3500. These heavy-duty trucks get the job done, and they do it in style. But which should you choose? Our comparison guide takes a closer look at these modern American powerhouses.
The heavy-duty 2019 Ford F-350 and Ram 3500 trucks are more than super powerful—they're thoroughly modern. Both trucks offer an almost overwhelming amount of configurations and options, though, which can make choosing between them somewhat complicated. Luckily, our comparison guide is here with the answers you seek.
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4 listings starting at $24,010


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Enterprise security has undergone a complete transformation as a result of rapid and sweeping changes that are remaking the way public sector organizations do business. New technologies and trends are changing the very definition of an enterprise, how and where we work, and the tools we use to do our jobs. These trends include -
- Mobility
- Cloud Applications
- Social Media


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Mount a network shared drive on GNU/Linux | Tutorial
Mount a network shared drive on GNU/Linux | Tutorial
In this post we will go through the process on how to mount a network shared drive on GNU/Linux. With this method, not only you can mount a network shared drive, you can also use this method to mount other network shared locations(network shared folders or other network shares). I have a network shared storage server, the server has HDDs in RAID configuration and the storage drives are shared over the network, I am going to use this setup as an example. Also in this post, we will cover the steps how to mount the network drives permanently and to mount automatically on boot.
There are couple of things we need to setup in order to make this configuration. We need to install an app called cifs-utils and we need to create a mount point.
Bellow you can find a video tutorial as well:
The process
Creating a mount point
First we need to create our mount point for our network share and for that we need to use the terminal. We will create the mount point in the /mnt folder. Start the terminal and use the following command:
sudo mkdir /mnt
sudo mkdir /mnt/share
Installing cifs-utils
Next what we need to do is to install the cifs-utils utility. This software will helps to mount and manage our network shares. Another reason we're going to use this software is because this software supports other non-Linux partition extensions, such as partitions which Windows use.
To install the software, use the command:
For apt package manager:
sudo apt install cifs-utils
For RPM package manager:
yum install cifs-utils
Mounting the network share
Now we can mount the network shared drive. First, please note, the commands bellow are an example and the parameters which are used are for my network setup. They will not be same as how is your network configured. You need to apply parameters according to your network settings.
First we use the command if credentials are set to the network share(meaning, the username and password is required to access the network share):
sudo mount.cifs //10.0.1.4/share /mnt/share/nmshare/ -o user=share,pass=share
Command description:
- sudo mount.cifs – To mount the network share using cifs software and with root privileges.
- //10.0.1.4/share – IP address and the name of the shared folder(this you need to change and to match your network settings)
- /mnt/share/nmshare/ – The mount point we created earlier where our network share will be placed.
- -o user=share,pass=share – Adding option to access the network share with credentials(adding username and password). Also, you can add option like this : -o username=share,password=share
If for your network share does not have credentials to access it, then you use the command to publicly mount the network share:
sudo mount.cifs //10.0.1.4/share /mnt/share/nmshare/
Mount permanently and automatically on boot
In order to make this configuration we need to access and edit the fstab config file. I kindly advise to backup the fstab config file first before making any changes to it. For this, we need to use the nano text editor and access the fstab file.
Use the command:
sudo -s nano /etc/fstab
You need to get a windows like this. At the bottom of the editor add the following line(again the parameters needs to match your network settings):
//10.0.1.4/share /mnt/share/nmshare cifs username=share,password=share 0 0
This is an example if you have credentials set to access your network share. To mount the network share which does not have credentials, use this command:
//10.0.1.4/share /mnt/share/nmshare cifs guest,uid=1000 0 0
Once you finish with editing the file, save and exit.


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Fly High [Postponed]
All | Workshops | Holiday Programmes | Exhibitions | Theatre | Nature | Outings | Open Houses
Events for 27 February 2020
Day Navigation
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The Curious Sky – Galaxy Edition
The Curious Sky is a multi-sensory arts play space inspired by elements of the weather.Find out more »
Seekor Singa, Seorang Putera & Sebingkai Cermin: Reflecting & Refracting Singapura at Malay Heritage Centre
Seekor Singa, Seorang Putera & Sebingkai Cermin: Reflecting & Refracting Singapura shines the spotlight on the legends of Singapore. With more than 120 exhibits on display, the special exhibition at Malay Heritage Centre examines Singapore's place and history all the way back to the 15th century. Using maps, artefacts and historical accounts, the Seekor Singa,Find out more »
Scholarly Treasures
Step into the shoes of a Chinese scholar, and have fun with the various artistic skills they had to pursue! Learn more about the brush, seal, paper, ink, and inkstone, and create your own Chinese hanging scroll artwork.Find out more »
Living Worlds: An Animal Planet Experience – Take a Step Behind the Screen at Science Centre Singapore
If you have ever watched an Animal Planet documentary and wondered how they captured such amazing footage, Living Worlds: An Animal Planet Experience at Science Centre Singapore's Annexe will certainly attract your interest. Living Worlds at Science Centre Singapore The exhibition at Science Centre Singapore's Annexe, which runs till March 2020, explores the interdependent relationshipFind out more »
2219: Futures Imagined at ArtScience Museum
Step into 2219: Futures Imagined and explore how our world might change over the next 200 years in a major exhibition developed by ArtScience Museum, as part of the Singapore Bicentennial commemoration. This immersive and experiential exhibition is a journey into imaginary future worlds, featuring over two dozen artists, architects, filmmakers, writers and theatre companies fromFind out more »
DAS Donate to Educate Pledge 2020
This lunar new year, the Dyslexia Association of Singapore (DAS) is calling out to everyone to contribute to the DAS Bursary Fund to help students from low-income families with dyslexia access specialist support at our learning centres.Find out more »
PLAYtime! Fatimah and Her Magic Socks
Dance, laugh, sing and imagine with Fatimah in this colourful theatrical delight especially created for children aged 2 to 6.Find out more »
Hands-on Exhibition: I-Opener
Come explore I-Opener, Playeum's sixth hands-on exhibition athe Children's Centre for Creativity. Designed for children aged 1-12, I-Opener empowers all families to discover their creative potential and explore their senses through artful play.Find out more »
Disney: Magic of Animation
Fall in love again with your favourite films and stories from Walt Disney Animation Studios! From Mickey Mouse to Anna and Elsa, uncover the magic behind almost a century of artistry and technological innovations that has brought to life our most beloved animated characters.Find out more »
Orchids of the Amazon
Exotic epiphytic orchids from the faraway Amazon region are brought closer to Singapore in Cloud Forest's Orchids of the Amazon, where 50 varieties are showcased amidst a lush green landscape of ferns, aroids and bromeliads that bring to mind the dense foliage of the rainforest.Find out more »
Gateway Kids Club
Gateway Theatre's weekly kids programme within the Gateway Theatre Creative Arts Spaces includes storytelling, arts and crafts, free play activities, as well as featured parenting talks and workshops.Find out more »
Drop-in Activity: Pop-up Cities
Inspired by architecture and art in the exhibition Suddenly Turning Visible: Art and Architecture in Southeast Asia (1969–1989), create buildings designed to pop out of a card!Find out more »
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An internet analytics is tool that makes use of the power of heat map modern technology to offer you a visual photo of exactly what site visitors are doing on your websites. A warm map is a graphical representation of information where the individual values consisted of in a matrix are represented as colors. Crazy egg reveals you where people are renovating their computer mouse on the page and where they click. So, this kind of tracking helps you recognize just what locations are catching one of the most focus and also communication from customers. Its "Scrollmap" function tells you where visitors desert the web page as well as where you need to add components to maintain them engaged. An additional feature called "Confetti" identifies clicks based on classifications like recommendation sources as well as search terms. In addition, this fantastic internet analytics device likewise provides Search Engine Optimization update tracking. After creating an internet site it becomes important for a freelance web developer to measure the efficiency of an internet site.
Web site owners as well as webmasters across the world would unanimously settle on the manifold benefits provided by internet analytics. Extracting the best out of such a program calls for inside-out knowledge concerning it. Irrespective of whether you make use of Kiss metrics or Google Analytics, you must have complete understanding of these devices and their functionalities. Every company has a website as well as every web site has a great deal of third party tags on the websites. These tags are used to track business which would assist make the decisions on business optimization. There are ton of various analytics software packages that transform the raw web information like clicks, hits, page sights to significant graphs, graphs, records and sectors. These records can be made use of to attain organization goals. There are innumerable tools and also resources readily available online which allows you check that the number of visitors has actually seen your web site, which showcases or web pages are most preferred.
Internet analytics is a procedure of gathering & gauging information, recognizing internet site visitor activity as well as actions, and enhancing the internet marketing process to make best use of ROI. If you want specific interpretation after that right here is one from Wikipedia – Internet analytics is the research of online habits in order to enhance it. Web analytics allows you to determine client engagement by measuring the site bounce price. Jump rate is a metrics that informs you the number of your site visitors visited just one web page on your website before bailing out. Internet analytics also lets you see the internet browser type, operating system and display resolution of the site visitors. This info allows you to supply enhanced individual experience and also give easy to use web pages. They assist you in tracking your website web traffic, condition, site visitors' requirements and actions.
Analytics is the missing web link in tracking the campaign to its core. It provides the website owners specified insight on how reliable their online as well as offline marketing campaigns are in encouraging their internet site visitors to take desired action. Internet analytics also supplies critical info made use of making strategic advertising choices. It's very simple to layout and publish website these days. There is no limitation to readymade layouts, easy to use HTML editors, and also low-cost developers. The volume of net target market is additionally growing rapidly as well as it's coming to be very essential to recognize your internet audience so as to get their attention to your product and services. If your web site is able to "fix" their problem swiftly, economically, and also conveniently they will become your long term consumer. Internet analytics aids you see your internet visitor's site navigating course.


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Property Reference: M539604
Short walk into the city centre | Private gated parking and also just a few metres from the station | Award winning farmers market and restaurant opposite
2 nights - mostly start on Fri or Sat
3 nights - must start on Fri
4 nights - must start on Mon
5 nights - 22nd or 29th Dec 2012 only.
"Located in a charming red brick former merchants store this comfortable apartment has everything you could want for a City break or a longer stay with a large and sociable open plan living space two bedrooms and two bathrooms.\nIf travelling by train the apartment is just a few metres walk from the station and if by car there is a private gated car park.\nJust 200 metres away is a convenient supermarket for stocking up on self catering supplies or if you wish to eat out then don't miss The Goods Shed opposite the apartment. It is a fabulous farmer's market cafe and restaurant serving organic and home cooked foods - excellent for breakfast before exploring the City Centre.\nA short walk will take you to the West Gate medieval City Walls entering through the iconic Westgate towers. From here you have a wealth of history culture entertainment and refreshments including the historic Cathedral which you can see from the apartment the Marlowe Theatre the Curzon Cinema and much much more to make your visit truly memorable."
No. 5 Merchants Store: sleeps 4 in 2 bedrooms and 2 bathrooms (of which 1 is en-suite). King 1, Double 1. Private gated parking for one car. Steps to the apartment block front door
Ground FloorHallwayOpen plan sitting/dining room and kitchenSofas seating 4 guests, dining table and chairs for 4 guests, Smart TVKitchen with electric oven and gas hob, dishwasher, fridge/freezer, microwaveUtility cupboard with washer/dryerMaster Bedroom (sleeps 2) Kingsize bed, en-suite bathroom with bath, handheld shower, WC and basinBedroom 2 (sleeps 2) double bedShower room with shower cubicle, WC and basin
OutsidePrivate gated car park with parking for one car
Private gated parking for one car A cot and highchair are available upon request. Guests are advised to bring their own bedding for the cot Accessibility - the apartment is on one level but there are a few steps to the front door No pets are permitted at No 5 Merchant Store Wifi Central heating
The entire City of Canterbury is on your doorstep - history, culture, shopping and more.
Very comfortable apartment, near to the centre. Would definitely recommend it to others
Very comfortable apartment, near to the centre. Would definitely recommend it to others
Published on: 2019-09-16


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Lipodisks, planar lipid bilayer structures stabilized by PEG-ylated lipids, were in the present study covalently bound and immobilized onto sensors for quartz crystal microbalance with dissipation monitoring (QCM-D) studies. It is shown that the modified sensors can be used to characterize the interaction of lipodisks with α-helical amphiphilic peptides with an accuracy similar to that obtained with well established fluorimetric approximations. The method presented has the great advantage that it can be used with peptides in their native form even if no fluorescent residues are present. The potential of the method is illustrated by determining the parameters describing the association of melittin, mastoparan X, and mastoparan with immobilized lipodisks. Both thermodynamic and kinetic analyses are possible. The presented method constitutes a useful tool for fundamental studies of peptide–membrane interactions and can also be applied to optimize the design of lipodisks, for example, for sustained release of antimicrobial peptides in therapeutic applications.
Metabolomic fingerprinting is a growing strategy for characterizing complex biological samples without detailed prior knowledge about the metabolic system. A two-way analysis system with liquid separation and mass spectrometric detection provides detail-rich data suitable for such fingerprints. As a model study, human urine samples, obtained after intake of coffee, tea, or water, were analyzed with capillary electrophoresis electrospray ionization time-of-flight mass spectrometry (CE−ESI-TOF-MS). In-house-developed software (in Matlab) was utilized to manage and explore the large amount of data acquired (230 CE−MS runs, each with 50−100 million nonzero data points). After baseline and noise reduction, followed by suitable binning in time and m/z, the data sets comprised 9 and 14 million data points in negative and positive ESI mode, respectively. Finally, a signal threshold was applied, further reducing the number to about 100 000 data points per data set. A set of interactive exploratory tools, utilizing principal component analysis (PCA) and analysis of variance (ANOVA) results based on a general linear model, facilitated visual interpretation with score plots (for group assessment) and differential fingerprints (for "hot spot" detection). In the model study highly significant differences due to beverage intake were obtained among the 10 first principal components (p < 10−6 for two of the components in both ESI modes). Especially, the contrasts between "coffee" and "tea or water" indicated several "hot spots" with highly elevated intensities (e.g., for uncharged masses 93, 94, 109, 119, 123, 132, 148, 169, 178, 187, 190, and 193) suitable for further analysis, for example, with tandem MS.
Flow control is central to microfluidics and chromatography. With decreasing dimensions and high pressures, precise fluid flows are often needed. In this paper, a high-pressure flow control system is presented, allowing for the miniaturization of chromatographic systems and the increased performance of microfluidic setups by controlling flow, composition and relative permittivity of two-component flows with CO2. The system consists of four chips: two flow actuator chips, one mixing chip and one relative permittivity sensor. The actuator chips, throttling the flow, required no moving parts as they instead relied on internal heaters to change the fluid resistance. This allows for flow control using miniaturized fluid delivery systems containing only a single pump or pressure source. Mobile phase gradients between 49% to 74% methanol in CO2 were demonstrated. Depending on how the actuator chips were dimensioned, the position of this range could be set for different method-specific needs. With the microfluidic control board, both flow and composition could be controlled from constant pressure sources, drift could be removed, and variations in composition could be lowered by 84%, resulting in microflows of CO2 and methanol with a variation in the composition of 0.30%.
The tracer-pulse method was extended and validated for the determination of multicomponent adsorption isotherms in liquid chromatography. Competitive adsorption isotherms can be determined for any number of solutes, up to the column resolution limit. The basic principle is to equilibrate the column with an eluent containing a mixture of the solutes and then measure the migration velocity of each of them through the column. It is easy to calculate the stationary phase concentrations from these velocities, given the eluent composition. As in frontal analysis, real competitive isotherm data are measured using this method, unlike other methods, which only produce parametric estimates. The method was used to measure the binary isotherms of beta-blockers on a Kromasil C8 column. The data were fitted to competitive bi-Langmuir adsorption isotherm functions and was found to agree well with the results of frontal analysis and the perturbation method. Computer simulations based on the isotherm parameters were performed and displayed very good agreement with the experimental chromatograms. An intriguing and seemingly paradoxical property is visualized and discussed: the fact that the injected molecules are not found in the detected peaks.
A recent proteomics-grade (95%+ sequence reliability) high-throughput de novo sequencing method utilizes the benefits of high resolution, high mass accuracy, and the use of two complementary fragmentation techniques collision-activated dissociation (CAD) and electron capture dissociation (ECD). With this high-fidelity sequencing approach, hundreds of peptides can be sequenced de novo in a single LC-MS/MS experiment. The high productivity of the new analysis technique has revealed a new bottleneck which occurs in data representation. Here we suggest a new method of data analysis and visualization that presents a comprehensive picture of the peptide content including relative abundances and grouping into families. The 2D mass mapping consists of putting the molecular masses onto a two-dimensional bubble plot, with the relative monoisotopic mass defect and isotopic shift being the axes and with the bubble area proportional to the peptide abundance. Peptides belonging to the same family form a compact group on such a plot, so that the family identity can in many cases be determined from the molecular mass alone. The performance of the method is demonstrated on the high-throughput analysis of skin secretion from three frogs, Rana ridibunda, Rana arvalis, and Rana temporaria. Two dimensional mass maps simplify the task of global comparison between the species and make obvious the similarities and differences in the peptide contents that are obscure in traditional data presentation methods. Even biological activity of the peptide can sometimes be inferred from its position on the plot. Two dimensional mass mapping is a general method applicable to any complex mixture, peptide and nonpeptide alike.
Capillary electrophoresis with laser-induced fluorescence detection is used to analyze 10 microL samples of human cerebrospinal fluid. Primary amine-containing compounds in untreated cerebrospinal fluid are labeled with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde prior to analysis, producing fluorescent isoindoles. Electropherograms containing approximately 50 peaks are obtained in less than 35 min from cerebrospinal fluid samples. Ten peaks in the electropherograms have been identified and quantitated: arginine, glutamine, threonine, valine, gamma-amino-n-butyric acid, serine, alanine, glycine, glutamic acid, and aspartic acid. Detection limits for these 10 amino acids range from 0.29 nM for gamma-amino-n-butyric acid to 100 nM for threonine, and separation efficiencies as high as 190,000 theoretical plates are obtained for these analytes. Electropherograms of cerebrospinal fluid samples from patients with Alzheimer's disease and from children with different neurological disorders are compared to those of healthy controls. Differences in individual amino acid levels are observed between the patient groups, and these differences appear to be disease and age related. These results indicate that analysis of cerebrospinal fluid by capillary electrophoresis will be useful as a selective, rapid, and sensitive tool for both diagnosis of central nervous system disorders and for study of the mechanisms of these disorders.
The poly(dimethylsiloxane) (PDMS) material provides a number of advantageous features, such as flexibility, elasticity, and transparency, making it useful in integrated analytical systems. Hard fused-silica capillary structures and soft PDMS channels can easily be combined by a tight fit, which offers many alternatives for structure combinations. PDMS and fused silica are in different ways prone to adsorption of low levels of organic compounds. The need for modification of the inner wall surface of PDMS channels may often be necessary, and in this paper, we describe an easy and effective method using the amine-containing polymer PolyE-323 to deactivate both fused-silica and PDMS surfaces. The adsorption of selected peptides to untreated surfaces was compared to PolyE-323-modified surfaces, using both radionuclide imaging and capillary electrophoresis experiments. The polyamine modification displayed a substantially reduced adsorption of three hydrophobic test peptides compared to the native PDMS surface. Filling and storage of aqueous solution were also possible in PolyE-323-modified PDMS channels. In addition, hybrid microstructures of fused silica and PDMS could simultaneously be deactivated in one simple coating procedure.
An interface in elastomeric poly(dimethylsiloxane) (PDMS) for on-line orthogonal coupling of packed capillary liquid chromatography (LC) (i.d. = 0.2 mm) with capillary electrophoresis (CE) in combination with sheathless electrospray ionization (ESI) time-of-flight mass spectrometric (TOFMS) detection is presented. The new interface has a two-level design, which in combination with a continuous CE electrolyte flow through the interface provides integrity of the LC effluent and the CE separation until an injection is desired. The transparent and flexible PDMS material was found to have a number of advantages when combined with fused silica column technology, including ease to follow the process and ease to exchange columns. By combining conventional microscale systems of LC, CE, and ESI−MS, respectively, the time scales of the individual dimensions were harmonized for optimal peak capacity per unit time. The performance of the LC−CE−TOFMS system was evaluated using peptides as model substances. A S/N of about 330 was achieved for leucine-enkephaline from a 0.5 μL LC injection of 25 μg/mL peptide standard.
We describe a simple and straightforward approach for homogeneous and isothermal detection of individual rolling circle replication (RCR) products, which represent individual padlock probe circularization events. The RCR products constitute tens of kilobases long single-stranded tandem repeated copies of the probe sequence, and in solution, they fold into micrometer-sized random coils. The method is based on the local enrichment of fluorescence-labeled probes that hybridize to the coiled RCR products compared to the concentration of free probes in solution. We present a detailed characterization of the fluorescence-labeled products using a highly sensitive and fast microscopy setup. At a 10(4)-fold excess of free label, we were able to detect and follow individual RCR products at a signal-to-background noise ratio of 27. This high signal-to-background noise ratio leaves room for analysis in a simple detection device at higher speeds or at lower labeling ratios.
Cellular receptor activity is often controlled through complex mechanisms involving interactions with multiple molecules, which can be soluble ligands and/or other cell surface molecules. In this study, we combine a fluorescence-based technology for real-time interaction analysis with fluorescence quenching to create a novel time-resolved proximity assay to study protein-receptor interactions on living cells. This assay extracts the binding kinetics and affinity for two proteins if they bind in proximity on the cell surface. One application of real-time proximity interaction analysis is to study relative levels of receptor dimerization. The method was primarily evaluated using the HER2 binding antibodies Trastuzumab and Pertuzumab and two EGFR binding antibodies including Cetuximab. Using Cetuximab and Trastuzumab, proximity of EGFR and HER2 was investigated before and after treatment of cells with the tyrosine-kinase inhibitor Gefitinib. Treated cells displayed 50% increased proximity signal, whereas the binding characteristics of the two antibodies were not significantly affected, implying an increase in the EGFR-HER2 dimer level. These results demonstrate that real-time proximity interaction analysis enables determination of the interaction rate constants and affinity of two ligands while simultaneously quantifying their relative colocalization on living cells.
A systematic X-ray absorption study at actinide N-6,(7) (4f -> 6d transitions) edges was performed for light-actinide oxides including data obtained for the first time for NpO2, PuO2 and UO3. The measurements were supported by ab initio calculations based on local-density approximation. with added 5f-5f Coulomb interaction (LDA+U). Improved energy resolution compared to common experiments at actinide L-2,L-3 (2p -> 6d transitions) edges allowed us to resolve the major structures of the 13 unoccupied 6d density of states (DOS) and estimate the crystal-field 116) splittings in the 6d shell directly from the spectra of light-actinide dioxides. The measurements demonstrated an enhanced sensitivity of the N-6,N-7, spectral shape to changes in the compound crystal structure. For nonstoichiometric NpO2-x, the filling of the entire band gap with Np 6d states was observed thus supporting a phase coexistence of Np metal and stoichiometric NpO2 which is in agreement with the tentative Np-O phase diagram.
The study addresses the possibilities of immobilizing the mobile species of actinides in the geosphere using metallic iron. Sorption on corroding iron is well-known, but there have been uncertainties with regard to the possibilities of reducing the actinyl species to sparingly soluble oxides and, thereby, permanently immobilizing them. Resonant inelastic X-ray scattering (RIXS) measurements at the actinide 5d edges on Fe foils exposed to uranium(VI) and neptunium(V) solutions in groundwater unambigiously indicate reduction of actinides to, respectively, uranium(IV) and neptunium(IV) on iron surfaces. The reduction manifests itself in an appearance of distinct specific signatures of uranium(IV) and neptunium(IV) in the RIXS profile of 5f-5f excitations. Such signatures and RIXS intensity/cross-section behavior with varying energy of incident photons can be reproduced by model atomic-multiplet calculations of the RIXS spectra. By normalizing the RIXS signal of corresponding 5f-5f excitations to core-to-core 6p-to-5d characteristic fluorescence transitions of actinides, their reduction rates on Fe samples with different exposure to actinide solutions can be estimated. Observed reduction implies similar processes in the nuclear waste canister thus suggesting reduced probability of nuclear waste release with ground waters from the canister.
Electric response to pH variations is employed to investigate Si nanoribbon field-effect transistors (SiNRFETs) operating in electrolyte with different gate configurations. For devices with a concluding gate electrode for direct metal electrolyte contact, a well-defined electrode reaction leading to a stable electrode potential is essential for retaining a stable electrical potential of the electrolyte. However, noble metals such as Pt do not meet the stability requirement and consequently bring severe distortions to the electronic response. For devices with an insulated gate electrode relying on the principle of capacitive gate coupling, the capacitance between the gate electrode and the electrolyte should be made much larger than the gate capacitance established between the SiNR and the electrolyte. In this case, an efficient gate control as well as a high stability against external disturbances can be ensured. Further studies show that surface charging of the gate insulator is the main cause responsible for the pH response of the SiNRFETs. Hence, devices with different gate configurations give rise to a comparable pH sensitivity.
A microchip in poly(dimethylsiloxane) (PDMS) for in-line solid-phase extraction-capillary electrophoresis-electrospray ionization-time-of-flight mass spectrometry (SPE-CE-ESI-TOF-MS) has been developed and evaluated. The chip was fabricated in a novel one-step procedure where mixed PDMS was cast over steel wires in a mold. The removed wires defined 50-um cylindrical channels. Fused-silica capillaries were inserted into the structure in a tight fit connection. The inner walls of the inserted fused-silica capillaries and the PDMS microchip channels were modified with a positively charged polymer, PolyE-323. The chip was fabricated in a two-level cross design. The channel at the lower level was packed with 5-um hyper-cross-linked polystyrene beads acting as a SPE medium used for desalting. The upper level channel acted as a CE channel and ended in an integrated emitter tip coated with conducting graphite powder to facilitate the electrical contact for sheathless ESI. An overpressure continuously provided fresh CE electrolyte independently of the flows in the different levels. Further studies were carried out in order to investigate the electrophoretic and flow rate properties of the chip. Finally, six-peptide mixtures, in different concentrations, dissolved in physiological salt solution was injected, desalted, separated, and sprayed into the mass spectrometer for analysis with a limit of detection in femtomole levels.
There is growing interest in cerebral microdialysis (MD) for sampling of protein biomarkers in neurointensive care (NIC) patients. Published data point to inherent problems with this methodology including protein interaction and biofouling leading to unstable catheter performance. This study tested the in vivo performance of a refined MD method including catheter surface modification, for protein biomarker sampling in a clinically relevant porcine brain injury model. Seven pigs of both sexes (10-12 weeks old; 22.2-27.3 kg) were included. Mean arterial blood pressure, heart rate, intracranial pressure (ICP) and cerebral perfusion pressure was recorded during the stepwise elevation of intracranial pressure by inflation of an epidural balloon catheter with saline (1 mL/20 min) until brain death. One naïve MD catheter and one surface modified with Pluronic F-127 (10 mm membrane, 100 kDa molecular weight cutoff MD catheter) were inserted into the right frontal cortex and perfused with mock CSF with 3% Dextran 500 at a flow rate of 1.0 μL/min and 20 min sample collection. Naïve catheters showed unstable fluid recovery, sensitive to ICP changes, which was significantly stabilized by surface modification. Three of seven naïve catheters failed to deliver a stable fluid recovery. MD levels of glucose, lactate, pyruvate, glutamate, glycerol and urea measured enzymatically showed an expected gradual ischemic and cellular distress response to the intervention without differences between naïve and surface modified catheters. The 17 most common proteins quantified by iTRAQ and nanoflow LC-MS/MS were used as biomarker models. These proteins showed a significantly more homogeneous response to the ICP intervention in surface modified compared to naïve MD catheters with improved extraction efficiency for most of the proteins. The refined MD method appears to improve the accuracy and precision of protein biomarker sampling in the NIC setting.
There is growing interest in sampling of protein biomarkers from the interstitial compartment of the brain and other organs using high molecular cutoff membrane microdialysis (MD) catheters. However, recent data suggest that protein sampling across such MD membranes is a highly complex process that needs to be further studied. Here, we report three major improvements for microdialysis sampling of proteins in complex biological matrixes. The improvements in this in vitro study using human ventricular cerebrospinal fluid as the sample matrix include increased fluid recovery control, decreased protein adsorption on the microdialysis membrane and materials, and novel quantitative mass spectrometry analysis. Dextrans in different concentrations and sizes were added to the perfusion fluid. It was found that dextrans with molecular mass 250 and 500 kDa provided a fluid recovery close to 100%. An improved fluid recovery precision could be obtained by self-assembly triblock polymer surface modification of the MD catheters. The modified catheters also delivered a significantly increased extraction efficiency for some of the investigated proteins. The final improvement was to analyze the dialysates with isobaric tagged (iTRAQ) proteomics, followed by tandem mass spectrometric analysis. By using this technique, 48 proteins could be quantified and analyzed with respect to their extraction efficiencies. The novel aspects of microdialysis protein sampling, detection, and quantification in biological fluids presented in this study should be considered as a first step toward better understanding and handling of the challenges associated with microdialysis sampling of proteins. The next step is to optimize the developed methodology in vivo.
The structures of organic phosphorous (P) compounds in aquatic sediments are to a large extent unknown although these compounds are considered to play an important role in regulating lake trophic status. To enhance identification of these compounds, a liquid chromatography (LC) method for their separation was developed. The stationary phase was porous graphitic carbon (PGC), and the mobile phases used in the gradient elution were compatible with both inductive coupled plasma atomic emission spectroscopy (ICP-AES) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). With LC-ICP-AES, eight different P containing peaks could be observed in the P chromatogram indicating that at least eight different P compounds were separated. With the setup of an information dependent acquisition (IDA) with ESI-MS/MS, the mass over charge (m/z) of compounds containing a phosphate group (H2PO3−, m/z 97) could be measured and further fragmentation experiments gave additional information on the structure of almost 40 separated P compounds, several were verified to be nucleotides. ICP-AES was very suitable in the development of the LC method and allowed screening and quantification of P compounds. The presented LC-ESI-MS/MS technique was able to identify several sediment organic P compounds.
We demonstrate detection of DNA coils formed from a Vibrio cholerae DNA target at picomolar concentrations using a novel optomagnetic approach exploiting the dynamic behavior and optical anisotropy of magnetic nanobead (MNB) assemblies. We establish that the complex second harmonic optical transmission spectra of MNB suspensions measured upon application of a weak uniaxial AC magnetic field correlate well with the rotation dynamics of the individual MNBs. Adding a target analyte to the solution leads to the formation of permanent MNB clusters, namely, to the suppression of the dynamic MNB behavior. We prove that the optical transmission spectra are highly sensitive to the formation of permanent MNB clusters and, thereby to the target analyte concentration. As a specific clinically relevant diagnostic case, we detect DNA coils formed via padlock probe recognition and isothermal rolling circle amplification and benchmark against a commercial equipment. The results demonstrate the fast optomagnetic readout of rolling circle products from bacterial DNA utilizing the dynamic properties of MNBs in a miniaturized and low-cost platform requiring only a transparent window in the chip.
The methodology described in this article will significantly reduce the time required for understanding the relations between chromatographic data and bioactivity assays. The methodology is a hybrid of hypothesis-based and data driven scientific approaches. In this work, a novel chromatographic data segmentation method is proposed, which demonstrates the capability of finding what volatile substances are responsible for antiviral and cytotoxic effects in the medicinal plant extracts. Up until now, the full potential of the separation methods has not been exploited in the life sciences. This was due to the lack of data ordering methods capable of adequately preparing the chromatographic information. Furthermore, the data analysis methods suffer from multidimensionality, requiring a large number of investigated data points. A new method is described for processing any chromatographic information into a vector. The obtained vectors of highly complex and different origin samples can be compared mathematically. The proposed method, efficient with relatively small sized data sets, does not suffer from multidimensionality. In this novel analytical approach, the samples did not need fractionation and purification, which is typically used in hypothesis-based scientific research. All investigations were performed using crude extracts possessing hundreds of phyto-substances. The antiviral properties of medicinal plant extracts were investigated using gas chromatography-mass spectrometry, antiviral tests, and proposed data analysis methods. The findings suggested that (i) beta-cis-caryophyllene, linalool, and eucalyptol possess antiviral activity, while (ii) thujones do not, and (iii) alpha-thujone, beta-thujone, cis-p-menthan-3-one, and estragole show cytotoxic effects.
Liquid extraction mass spectrometry imaging (MSI) experiments provide users with direct analysis of biological surfaces with minimal sample preparation. Until now, much of the effort to increase spatial resolution for MSI with liquid extraction techniques has focused on reducing the size of the sampling area. However, this can be experimentally challenging. Here, we present oversampling as a simple alternative to increase the spatial resolution using nanospray desorption electrospray ionization (nano-DESI) MSI. By imaging partial rat spinal cord tissue sections, two major concerns with oversampling are addressed: whether endogenous molecules are significantly depleted from repeated sampling events and whether analytes are redistributed as a result of oversampling. In depth examination of ion images for representative analytes show that depletion and redistribution do not affect analyte localization in the tissue sample. Nano-DESI MSI experiments using three times oversampling provided higher spatial resolution, allowing the observation of features not visible with undersampling. Although proper care must be taken to ensure that oversampling will work in specific applications, we envision oversampling as a simple approach to increase image quality for liquid extraction MSI techniques.
Prostaglandins (PG) are an important class of lipid biomolecules that are essential in many biological processes, including inflammation and successful pregnancy. Despite a high bioactivity, physiological concentrations are typically low, which makes direct mass spectrometric analysis of endogenous PG species challenging. Consequently, there have not been any studies investigating PG localization to specific morphological regions in tissue sections using mass spectrometry imaging (MSI) techniques. Herein, we show that silver ions, added to the solvent used for nanospray desorption electrospray ionization (nano-DESI) MSI, enhances the ionization of PGs and enables nano-DESI MSI of several species in uterine tissue from day 4 pregnant mice. It was found that detection of [PG + Ag](+) ions increased the sensitivity by similar to 30 times, when compared to [PG - H](-) ions. Further, the addition of isotopically labeled internal standards enabled generation of quantitative ion images for the detected PG species. Increased sensitivity and quantitative MSI enabled the first proof-of-principle results detailing PG localization in mouse uterus tissue sections. These results show that PG species primarily localized to cellular regions of the luminal epithelium and glandular epithelium in uterine tissue. Further, this study provides a unique scaffold for future studies investigating the PG distribution within biological tissue samples.
Capillary electrophoresis mass spectrometry (CE-MS) is an established technique for targeted and untargeted analysis of metabolites from complex biological samples. However, current CE-MS devices rely on liquid sample extracts, which restricts acquisition of spatially defined chemical information from tissue samples. The ability to chemically profile distinct cellular regions in tissue can contribute better understanding to molecular foundations in health and disease. Therefore, we describe the first CE-MS device capable of untargeted metabolite profiling directly from defined morphological regions of solid tissue sections. With surface sampling capillary electrophoresis mass spectrometry (SS-CE-MS), endogenous molecules are sampled and detected from a single defined tissue location. Characterization of SS-CE MS from different locations of the outer epidermal layer of A. Cepa demonstrated reproducible relative migration times and a peak area RSD of 20% (n = 5). Further, relative migration times were conserved for endogenous metabolites in tissues with varying complexities, including brain, spinal cord, and kidney. Results from proof-of-principle experiments from distinct morphological tissue regions reveal simultaneous analysis of small and large biomolecules, confident metabolite annotation, identification of in-source fragmentation interferences, and discrete isomeric abundances related to biological function. We envision that this new tool will provide in-depth chemical profiling and annotation of molecules in distinct cellular regions of tissue for improved biological understanding.
A novel on-target phosphopeptide enrichment method is presented that allows specific enrichment and direct analysis by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) of phosphorylated peptides. Spots consisting of a thin film of anatase titanium dioxide are sintered onto a conductive glass surface. Enrichment and analysis can be performed on the modified target with minimal sample handling. The protocol leads to an enrichment efficiency that is superior to what has been reported before for similar methods. The method was tested using beta-casein as a model phosphorylated protein as well as with a custom peptide mixed with its phosphorylated form. A very low detection limit, a significantly improved phosphoprofiling capability, and a simple experimental approach provide a powerful tool for the enrichment, detection, and analysis of phosphopeptides.
A simple method for on-target enrichment and subsequent separation and analysis of phosphorylated peptides is presented. The tryptic digest of a phosphorylated protein, in this case beta-casein, is loaded onto a spot on a thin stripe made of mesoporous TiO2 sintered onto a conductive glass surface. After washing with a salicylic buffer in order to remove the nonphosphorylated peptides, the stripe is placed in an elution chamber containing a phosphate solution. In a way analogous to thin layer chromatography (TLC), the phosphate solution acts as an eluent, clearly separating multi- and monophosphorylated peptides. By performing matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) along the stripe, the detection of all phosphorylated peptides present in the digest is facilitated, as they are isolated from each other. The method was also tested on commercial drinking milk, achieving successful separation between multi- and monophosphorylated peptides, as well as a detection limit in the femtomole range. As the enrichment, separation, and analysis take place in the same substrate, sample handling and risk of contamination and sample loss is minimized. The results obtained suggest that the method, once optimized, may successfully provide a complete phosphoproteome.
Techniques for manipulating, separating, and trapping particles and cells are highly desired in today's bioanalytical and biomedical field. The microfluidic chip-based acoustic noncontact trapping method earlier developed within the group now provides a flexible platform for performing cell- and particle-based assays in continuous flow microsystems. An acoustic standing wave is generated in etched glass channels (600x61 microm2) by miniature ultrasonic transducers (550x550x200 microm3). Particles or cells passing the transducer will be retained and levitated in the center of the channel without any contact with the channel walls. The maximum trapping force was calculated to be 430+/-135 pN by measuring the drag force exerted on a single particle levitated in the standing wave. The temperature increase in the channel was characterized by fluorescence measurements using rhodamine B, and levels of moderate temperature increase were noted. Neural stem cells were acoustically trapped and shown to be viable after 15 min. Further evidence of the mild cell handling conditions was demonstrated as yeast cells were successfully cultured for 6 h in the acoustic trap while being perfused by the cell medium at a flowrate of 1 microL/min. The acoustic microchip method facilitates trapping of single cells as well as larger cell clusters. The noncontact mode of cell handling is especially important when studies on nonadherent cells are performed, e.g., stem cells, yeast cells, or blood cells, as mechanical stress and surface interaction are minimized. The demonstrated acoustic trapping of cells and particles enables cell- or particle-based bioassays to be performed in a continuous flow format.
Miniaturization of analytical separation methods offers several advantages, including short run times, high resolution, and high recovery of the sample constituents. To optimize these parameters, the reversible adsorption (to minimize loss in resolution), as well as the irreversible adsorption (to minimize loss of analytes) must be quantified. However, no useful equation is available for the calculation of the variance of reversible adsorption. Therefore, we have taken another approach to quantify the reversible interaction. The method is unique and important since no equation for calculation of this variance is required. Instead, two experiments are required, which are run under such conditions that the variance of a certain parameter has the same numerical value in the two experiments (one with and without EOF), except for the variance of reversible adsorption. The approach is universal in the sense that it can be used for many Is different mathematical concepts and be modified to also cover certain functions other than a sum of parameters. We have also introduced a simple expression for the irreversible adsorption, which shows that the hydrophobic interaction from only two methyl groups in the coating gives rise to as much as 40-50% loss of protein, and the width of the zones in the capillary with this coating was 8-15% larger compared to the zone width in the polyacrylamide-coated capillaries. The reproducibility in migration time, peak area, and peak width in two consecutive runs in capillaries with two methyl groups in the coating was very low, but in EOF-free polyacrylamide-coated capillaries extremely high, indicating that the reversible and irreversible adsorption of proteins to this coating is negligible. The scanning detector, frequently used in free zone electrophoresis in the 1960s-1970s, gives true separation parameters and is, therefore, much preferable to the stationary detector used in most CE experiments, because this detector gives apparent separation parameters.
In this paper, we utilize bulk acoustic waves to control the position of microparticles inside droplets in two-phase microfluidic systems and demonstrate a method to enrich the microparticles. In droplet microfluidics, different unit operations are combined and integrated on-chip to miniaturize complex biochemical assays. We present a droplet unit operation capable of controlling the position of microparticles during a trident shaped droplet split. An acoustic standing wave field is generated in the microchannel, and the acoustic forces direct the encapsulated microparticles to the center of the droplets. The method is generic, requires no labeling of the microparticles, and is operated in a noncontact fashion. It was possible to achieve 2+-fold enrichment of polystyrene beads (5 μm in diameter) in the center daughter droplet with an average recovery of 89% of the beads. Red blood cells were also successfully manipulated inside droplets. These results show the possibility to use acoustophoresis in two-phase systems to enrich microparticles and open up the possibility for new droplet-based assays that are not performed today.
Single-molecule experimental techniques have recently shown to be of significant interest for use in numerous applications in both the research laboratory and industrial settings. Although many single-molecule techniques exist, the nanopore platform is perhaps one of the more popular techniques due to its ability to act as a molecular sensor of biological macromolecules. For example, nanopores offer a unique, new method for probing various properties of proteins and can contribute to elucidating key biophysical information in conjunction with existing techniques. In the present study, various forms of bovine serum albumin (BSA) are detected including thermally refolded BSA, urea-denatured BSA, and multiple forms of BSA detected at elevated electric field strengths (with and without urea). We also provide excluded volume measurements for each of these states that normally are difficult to obtain due to unknown and unstable protein conformations.
The present study examines pyruvate kinase-conjugated antibodies for potential use in EUSA applications. The conjugates had an acceptable stability, and the coupling inflicted only minor impairment on the kinase activity. To mimic the setup of an immunoassay under development, a test antigen (BSA) was attached to polystyrene nanoparticles. This arrangement was found to be suitable as solid support for presentation of antigens in sensitive bioluminescence assays. The nanoparticles were well characterized in terms of protein surface load and were used to establish the number of conjugate complexes needed to generate a detectable signal. Under the biochemical conditions employed here, the detection limit of the pyruvate kinase conjugate lies in the femtomole range.
Small neutral losses from charge-reduced species [M + nH]((n-1)+center dot) is one of the most abundant fragmentation channels in both electron capture dissociation, ECD, and electron transfer dissociation, ETD. Several groups have previously studied these losses on particular examples. Now, the availability of a large (11491 entries) SwedECD database (http://www.bmms.uu.se/CAD/indexECD.html) of high-resolution ECD data sets on doubly charged tryptic peptides has made possible a systematic study involving statistical evaluation of neutral losses from [M + 2H](+center dot) ions. Several new types of losses are discovered, and 16 specific (>94%) losses are characterized according to their specificity and sensitivity, as well as occurrence for peptides of different lengths. On average, there is more than one specific loss per ECD mass spectrum, and two-thirds of all MS/MS data sets in SwedECD contain at least one specific loss. Therefore, specific neutral losses are analytically useful for improved database searching and de novo sequencing. In particular, N and GG isomeric sequences can be distinguished. The pattern of neutral losses was found to be remarkably dissimilar with the losses from radical z(center dot) fragment ions: e.g., there is no direct formation of w ions from the reduced species. This finding emphasizes the difference in fragmentation behaviors of hydrogen-abundant and hydrogen-deficient species.
The distributions of positron emission tomography (PET) ligands in rat brain tissue sections were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). The detection of the PET ligands was possible following the use of a solvent-free dry MALDI matrix application method employing finely ground dry α-cyano-4-hydroxycinnamic acid (CHCA). The D2 dopamine receptor antagonist 3,5-dichloro-N-{[(2S)-1-ethylpyrrolidin-2-yl]methyl}-2-hydroxy-6-methoxybenzamide (raclopride) and the D1 dopamine receptor antagonist 7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol (SCH 23390) were both detected at decreasing abundance at increasing period postdosing. Confirmation of the compound identifications and distributions was achieved by a combination of mass-to-charge ratio accurate mass, isotope distribution, and MS/MS fragmentation imaging directly from tissue sections (performed using MALDI TOF/TOF, MALDI q-TOF, and 12T MALDI-FT-ICR mass spectrometers). Quantitative data was obtained by comparing signal abundances from tissues to those obtained from quantitation control spots of the target compound applied to adjacent vehicle control tissue sections (analyzed during the same experiment). Following a single intravenous dose of raclopride (7.5 mg/kg), an average tissue concentration of approximately 60 nM was detected compared to 15 nM when the drug was dosed at 2 mg/kg, indicating a linear response between dose and detected abundance. SCH 23390 was established to have an average tissue concentration of approximately 15 μM following a single intravenous dose at 5 mg/kg. Both target compounds were also detected in kidney tissue sections when employing the same MSI methodology. This study illustrates that a MSI may well be readily applied to PET ligand research development when using a solvent-free dry matrix coating.
An approach to sequence-dependent retention time prediction of peptides based on the concept of liquid chromatography at critical conditions (LCCC) is presented. Within the LCCC approach applied to biopolymers (BioLCCC), the specific retention time corresponds to a particular sequence. In combination with mass spectrometry, this approach provides an efficient tool to solve problems wherein the protein sequencing is essential. In this work, we present a theoretical background of the BioLCCC concept and demonstrate experimentally its feasibility for sequence-dependent LC retention time prediction for peptides. BioLCCC model is based on three notions: ( a) a random walk model for a macromolecule chain; (b) an entropy and energy compensation for the macromolecules within the adsorbent pore; and ( c) a set of phenomenological parameters for the effective interaction energies of interactions between the amino acid residues and the adsorbent surface. In this work, the phenomenological parameters have been obtained for C-18 reversed-phase HPLC. Note, that contrary to alternative additive models for retention time prediction based on summation of the so-called "retention coefficients", the BioLCCC approach takes into account the location of amino acids within the primary structure of a peptide and, thus, allows the identification of the peptides having the same composition of amino acids but differing by their arrangement. As a result, this new approach allows prediction of retention time for any possible amino acid sequence in particular HPLC experiments. In addition, the BioLCCC model lacks of main drawbacks of additive approaches that predict retention time for sequences of limited chain lengths and provide information about amino acid composition only. The proposed BioLCCC approach was characterized experimentally using LTQ FT LC-MS and LC-MS/MS data obtained earlier for Escherichia coli. The HPLC system calibration was performed using peptide retention standards. The results received show a linear correlation between predicted and experimental retention times, with a correlation coefficient, R-2, of 0.97 for a peptide standard mixture and 0.9 for E. coli data, respectively, with the standard error below 1 min. The work presents the first description of a BioLCCC approach for high-throughput peptide characterization and preliminary results of its feasibility tests.
This work presents sensitive detection of bacterial genomic DNA using a magnetic nanoparticle-based substrate-free method. For the first time, such a method is employed for detection of a clinically relevant analyte by implementing a solid-phase-based molecular probing and amplification protocol that can be executed in 80 min. The molecular detection and amplification protocol is presented and verified on samples containing purified genomic DNA from Escherichia coli cells, showing that as few as 50 bacteria can be detected. This study moves the use of volume-amplified magnetic nanoparticles one step further toward rapid, sensitive, and selective infectious diagnostics.
We investigated the application of the LTQ-Orbitrap mass spectrometer (LTQ-Velos Pro, Thermo Fisher) for resolving complex mixtures of natural aquatic dissolved organic matter (DOM) and compared this technique to the more established state-of-the-art technique, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS, Bruker Daltonics), in terms of the distribution of molecular masses detected and the reproducibility of the results collected. The Orbitrap was capable of excellent reproducibility: Bray-Curtis dissimilarity between duplicate measurements was 2.85 +/- 0.42% (mean +/- standard deviation). The Orbitrap was also capable of the detection of most major ionizable organic molecules in typical aquatic mixtures, with the exception of most sulfur and phosphorus containing masses. This result signifies that the Orbitrap is an appropriate technique for the investigation of very subtle biogeochemical processing of bulk DOM. The lower costs (purchase and maintenance) and wider availability of Orbitrap mass spectrometers in university departments means that the tools necessary for research into DOM processing at the molecular level should be accessible to a much wider group of scientists than before. The main disadvantage of the technique is that substantially fewer molecular formulas can be resolved from a complex mixture (roughly one third as many), meaning some loss of information. In balance, most biogeochemical studies that aim at molecularly fingerprinting the source of natural DOM could be satisfactorily carried out with Orbitrap mass spectrometry. For more targeted metabolomic studies where individual compounds are traced through natural systems, FTICR-MS remains advantageous.
Formalin-fixed and paraffin-embedded (FFPE) and optimal cutting temperature (OCT)-embedded and frozen tissue specimens in biobanks are highly valuable in clinical studies but proteomic and post-translational modification (PTM) studies using mass spectrometry (MS) have been limited due to structural arrangement of proteins and contaminations from embedding material. This study aims to develop a parallel proteomic workflow for FFPE and OCT/frozen samples that allows for large-scale, quick, reproducible, qualitative, and quantitative high-resolution MS analysis. The optimized protocol gives details on removal of embedding material, protein extraction, and multienzyme digestion using filter-aided sample preparation method. The method was evaluated by investigating the protein expression levels in nonmuscle-invasive and muscle-invasive bladder cancer samples in two cohorts and MS spectra were carefully reviewed for contaminations. More than 2000 and 3000 proteins in FFPE and OCT/frozen samples, respectively, were identified, and samples could be clustered in different tumor stages based on their protein expression. Furthermore, more than 250 and 400 phosphopeptides could be identified from specific patient samples of FFPE and OCT/frozen, respectively, using titanium dioxide enrichment. The paper presents unique data describing the similarities and differences observed in FFPE and OCT/frozen samples and shows the feasibility to detect proteins and site-specific phosphorylation even after long-term storage of clinical samples.
We developed a technique to monitor spatially confined surface reactions with mass spectrometry under ambient conditions, without the need for voltage or organic solvents. Fused-silica capillaries immersed in an aqueous solution, positioned in close proximity to each other and the functionalized surface, created a laminar flow junction with a resulting reaction volume of similar to 5 pL. The setup was operated with a syringe pump, delivering reagents to the surface through a fused silica capillary. The other fused-silica capillary was connected to a Venturi easy ambient sonic-spray ionization source, sampling the resulting analytes at a slightly higher flow rate compared to the feeding capillary. The combined effects of the inflow and outflow maintains a chemical microenvironment, where the rate of advective transport overcomes diffusion. We show proof-of-concept where acetylcholinesterase was immobilized on an organosiloxane polymer through electrostatic interactions. The hydrolysis of acetylcholine by acetylcholinesterase into choline was monitored in real-time for a range of acetylcholine concentrations, fused-silica capillary geometries, and operating flow rates. Higher reaction rates and conversion yields were observed with increasing acetylcholine concentrations, as would be expected.
Partially or fully disordered proteins are instrumental for signal-transduction pathways; however, many mechanistic aspects of these proteins are not well-understood. For example, the number and nature of intermediate states along the binding pathway is still a topic of intense debate. To shed light on the conformational heterogeneity of disordered protein domains and their complexes, we performed single-molecule experiments by translocating disordered proteins through a nanopore embedded within a thin dielectric membrane. This platform allows for single-molecule statistics to be generated without the need of fluorescent labels or other modification groups. These studies were performed on two different intrinsically disordered protein domains, a binding domain from activator of thyroid hormone and retinoid receptors (ACTR) and the nuclear coactivator binding domain of CREB-binding protein (NCBD), along with their bimolecular complex. Our results demonstrate that both ACTR and NCBD populate distinct conformations upon translocation through the nanopore. The folded complex of the two disordered domains, on the other hand, translocated as one conformation. Somewhat surprisingly, we found that NCBD undergoes a charge reversal under high salt concentrations. This was verified by both translocation statistics as well as by measuring the C-potential. Electrostatic interactions have been previously suggested to play a key role in the association of intrinsically disordered proteins, and the observed behavior adds further complexity to their binding reactions.
An acoustic microfluidic system for miniaturized fluorescence-activated cell sorting (mu FACS) is presented. By excitation of a miniaturized piezoelectric transducer at 10 MHz in the microfluidic channel bottom, an acoustic standing wave is formed in the channel. The acoustic radiation force acting on a density interface causes fluidic movement, and the particles or cells on either side of the fluid interface are displaced in a direction perpendicular to the standing wave direction. The small size of the transducer enables individual manipulation of cells passing the transducer surface. At constant transducer activation the system was shown to accomplish up to 700 mu m sideways displacement of 10 mu m beads in a 1 mm wide channel. This is much larger than if utilizing the acoustic radiation force acting directly on particles, where the limitation in maximum displacement is between a node and an antinode which at 10 MHz is 35 mu m. In the automatic sorting setup, the system was demonstrated to successfully sort single cells of E-GFP expressing beta-cells.
Grazing emission X-ray fluorescence (GEXRF) is well suited for nondestructive elemental-sensitive depth-profiling measurements on samples with nanometer-sized features. By varying the grazing emission angle under which the X-ray fluorescence signal is detected, the probed depth range can be tuned from a few to several hundred nanometers. The dependence of the XRF intensity on the grazing emission angle can be assessed in a sequence of measurements or in a scanning-free approach using a position-sensitive area detector. Hereafter, we will show that the combination of scanning-free GEXRF and fluorescence detected X-ray absorption spectroscopy (XAS) allows for depth-resolved chemical speciation measurements with nanometer-scale accuracy. While the conventional grazing emission geometry is advantageous to minimize self-absorption effects, the use of a scanning-free setup makes the sequential scanning of the grazing emission angles obsolete and paves the way toward time-resolved depth-sensitive XAS measurements. The presented experimental approach was applied to study the surface oxidation of an Fe layer on the top of bulk Si and of a Ge bulk sample. Thanks to the penetrating properties and the insensitivity toward the electric conduction properties of the incident and emitted X-rays, the presented experimental approach is well suited for in situ sample surface studies in the nanometer regime.
Imaging mass spectrometry (IMS) that utilizes matrix-assisted laser desorption/ionization (MALDI) technology can provide a molecular ex vivo view of resected organs or whole-body sections from an animal, making possible the label-free tracking of both endogenous and exogenous compounds with spatial resolution and molecular specificity. Drug distribution and, for the first time, individual metabolite distributions within whole-body tissue sections can be detected simultaneously at various time points following drug administration. IMS analysis of tissues from 8 mg/kg olanzapine dosed rats revealed temporal distribution of the drug and metabolites that correlate to previous quantitative whole-body autoradiography studies. Whole-body MALDI IMS is further extended to detecting proteins from organs present in a whole-body sagittal tissue section. This technology will significantly help advance the analysis of novel therapeutics and may provide deeper insight into therapeutic and toxicological processes, revealing at the molecular level the cause of efficacy or side effects often associated with drug administration.
The recent increase in extensively drug-resistant bacterial pathogens and the associated increase of morbidity and mortality demonstrate the immediate need for new antibiotic backbones with novel mechanisms of action. Here, we report the development of the PepSAVI-MS pipeline for bioactive peptide discovery. This highly versatile platform employs mass spectrometry and statistics to identify bioactive peptide targets from complex biological samples. We validate the use of this platform through the successful identification of known bioactive peptides from a botanical species, Viola odorata. Using this pipeline, we have widened the known antimicrobial spectrum for V. odorata cyclotides, including antibacterial activity of cycloviolacin O2 against A. baumannii. We further demonstrate the broad applicability of the platform through the identification of novel anticancer activities for cycloviolacins by their cytotoxicity against ovarian, breast, and prostate cancer cell lines.
An operando electrochemical quartz crystal microbalance with dissipation monitoring (EQCM-D) with simultaneous in situ electrochemical impedance spectroscopy (EIS) has been developed and applied to study the solid electrolyte interphase (SEI) formation on copper current collectors in Li-ion batteries. The findings are backed by EIS simulations and complementary analytical techniques, such as online electrochemical mass spectrometry (OEMS) and X-ray photoelectron spectroscopy (XPS). The evolution of mass and the mechanical properties of the SEI are directly correlated to the electrode impedance. Electrolyte reduction at the anode carbon active material initiates dissolution, diffusion, and deposition of reaction side products throughout the cell and increases electrolyte viscosity and the ohmic cell resistance as a result. On Cu the reduction of CuOx and HF occurs at >1.5 V and forms an initial LiF-rich interphase while electrolyte solvent reduction at <0.8 V vs Li+/Li adds a second, less rigid layer on top. Both the shear storage modulus and viscosity of the SEI generally increase upon cycling but-along with the SEI Li+ diffusion coefficient-also respond reversibly to electrode potential, likely as a result of Li+/EC interfacial concentration changes. Combined EIS-EQCM-D provides unique prospects for further studies of the highly dynamic structure-function relationships of electrode interphases in Li ion batteries.
Valence-to-core resonant inelastic X-ray scattering (RIXS) and high energy resolution fluorescence detection (HERFD) X-ray absorption measurements were performed at the U (L)3 edges of UO2 and UO2(NO3)(2)(H2O)(6). The results are compared with model calculations based on the local-density-approximation formalism, taking into account Coulomb interaction U (LDA + U). We show that despite strong 5f-5f electronic correlations in the studied systems and the use of core-level excitations in the intermediate stage of the spectroscopic process, the RIXS technique probes a convolution of the single-particle densities of states in the valence and conduction bands. For UO2, the detected crystal-field splitting between the U 6d e(g) and t(2g) orbitals from the RIXS spectra (similar to 3.5 eV) is larger than that previously derived from optical spectroscopy. Furthermore, by using an example of the U0.75Pu0.25O2 mixed oxide, we show that the RIXS technique at the U L-3 edges is sensitive to the substitution of U with other actinide, in contrast to conventional X-ray absorption methods. That is, due to changes in the occupied part rather than in the unoccupied part of the U 6d states caused by the substitution.
Advances in mass spectrometry imaging that improve both spatial and mass resolution are resulting in increasingly larger data files that are difficult to handle with current software. We have developed a novel near-lossless compression method with data entropy reduction that reduces the file size significantly. The reduction in data size can be set at four different levels (coarse, medium, fine, and superfine) prior to running the data compression. This can be applied to spectra or spectrum-by-spectrum, or it can be applied to transpose arrays or array-by-array, to efficiently read the data without decompressing the whole data set. The results show that a compression ratio of up to 5.9:1 was achieved for data from commercial mass spectrometry software programs and 55:1 for data from our in-house developed mslQuant program. Comparing the average signals from regions of interest, the maximum deviation was 0.2% between compressed and uncompressed data sets with coarse accuracy for the data entropy reduction. In addition, when accessing the compressed data by selecting a random m/z value using mslQuant, the time to update an image on the computer screen was only slightly increased from 92 (+/- 32) ms (uncompressed) to 114 (+/- 13) ms (compressed). Furthermore, the compressed data can be stored on readily accessible servers for data evaluation without further data reprocessing. We have developed a space efficient, direct access data compression algorithm for mass spectrometry imaging, which can be used for various data-demanding mass spectrometry imaging applications.
This paper presents msIQuant, a novel instrument- and manufacturer-independent quantitative mass spectrometry imaging software suite that uses the standardized open access data format imzML. Its data processing structure enables rapid image display and the analysis of very large data sets (>50 GB) without any data reduction. In addition, msIQuant provides many tools for image visualization including multiple interpolation methods, low intensity transparency display, and image fusion. It also has a quantitation function that automatically generates calibration standard curves from series of standards that can be used to determine the concentrations of specific analytes. Regions-of-interest in a tissue section can be analyzed based on a number of quantities including the number of pixels, average intensity, standard deviation of intensity, and median and quartile intensities. Moreover, the suite's export functions enable simplified postprocessing of data and report creation. We demonstrate its potential through several applications including the quantitation of small molecules such as drugs and neurotransmitters. The msIQuant suite is a powerful tool for accessing and evaluating very large data sets, quantifying drugs and endogenous compounds in tissue areas of interest, and for processing mass spectra and images.
Despite great interest, investments, and efforts, the ongoing search for plasma protein biomarkers for disease so far has come up surprisingly empty-handed. While discovery programs have revealed large numbers of biomarker candidates, the clinical utility has been validated for only a very small number of these. While this disappointing state of affairs may suggest that plasma protein biomarkers have little more to offer for diagnostics, we take the perspective that experimental conditions might not have been optimal, and that analyses will be required that offer far greater sensitivity than currently available, in terms of numbers of molecules needed for unambiguous detection. Accordingly, techniques are needed to search deep and wide for protein biomarker candidates. The requirements and feasibility of such assays will be discussed.
A wide variety of biological processes rely upon interactions between proteins and lipids, ranging from molecular transport to the organization of the cell membrane. It was recently established that electrospray ionization mass spectrometry (ESI-MS) is capable of capturing transient interactions between membrane proteins and their lipid environment, and a detailed understanding of the underlying processes is therefore of high importance. Here, we apply ESIMS to investigate the factors that govern complex formation in solution and gas phases by comparing nonselective lipid binding with soluble and membrane proteins. We find that exogenously added lipids did not bind to soluble proteins, suggesting that lipids have a low propensity to form electrospray ionization adducts. The presence of detergents at increasing micelle concentrations, on the other hand, resulted in moderate lipid binding to soluble proteins. A direct ESI-MS comparison of lipid binding to the soluble protein serum albumin and to the integral membrane protein NapA shows that soluble proteins acquire fewer lipid adducts. Our results suggest that protein lipid complexes form via contacts between proteins and mixed lipid/detergent micelles. For soluble proteins, these complexes arise from nonspecific contacts between the protein and detergent/lipid micelles in the electrospray droplet. For membrane proteins, lipids are incorporated into the surrounding micelle in solution, and complex formation occurs independently of the ESI process. We conclude that the lipids in the resulting complexes interact predominantly with sites located in the transmembrane segments, resulting in nativelike complexes that can be interrogated by MS.
Nanospray desorption electrospray ionization (nano-DESI) combined with tandem mass spectrometry (MS/MS), high-resolution mass analysis of the fragment ions (m/Δm = 17 500 at m/z 200), and rapid spectral acquisition enabled simultaneous imaging and identification of a large number of metabolites and lipids from 92 selected m/z windows (±1 Da) with a spatial resolution of better than 150 μm. Mouse uterine sections of implantation sites on day 6 of pregnancy were analyzed in the ambient environment without any sample pretreatment. MS/MS imaging was performed by scanning the sample under the nano-DESI probe at 10 μm/s, while higher-energy collision-induced dissociation (HCD) spectra were acquired for a targeted inclusion list of 92 m/z values at a rate of ∼6.3 spectra/s. Molecular ions and their corresponding fragments, separated by high-resolution mass analysis, were assigned on the basis of accurate mass measurement. Using this approach, we were able to identify and image both abundant and low-abundance isobaric and isomeric species within each m/z window. MS/MS analysis enabled efficient separation and identification of isomeric and isobaric phospholipids that are difficult to separate in full-scan mode. Furthermore, we identified several metabolites associated with early pregnancy and obtained the first 2D images of these molecules.
An automated platform has been developed for acquisition and visualization of mass spectrometry imaging (MSI) data using nanospray desorption electrospray ionization (nano-DESI). The new system enables robust operation of the nano-DESI imaging source over many hours by precisely controlling the distance between the sample and the nano-DESI probe. This is achieved by mounting the sample holder onto an automated XYZ stage, defining the tilt of the sample plane, and recalculating the vertical position of the stage at each point. This approach is useful for imaging of relatively flat samples such as thin tissue sections. Custom software called MSI QuickView was developed for visualization of large data sets generated in imaging experiments. MSI QuickView enables fast visualization of the imaging data during data acquisition and detailed processing after the entire image is acquired. The performance of the system is demonstrated by imaging rat brain tissue sections. Low background noise enables simultaneous detection of lipids and metabolites in the tissue section. High-resolution mass analysis combined with tandem mass spectometry (MS/MS) experiments enabled identification of the observed species. In addition, the high dynamic range (>2000) of the technique allowed us to generate ion images of low-abundance isobaric lipids. A high-spatial resolution image was acquired over a small region of the tissue section revealing the distribution of an abundant brain metabolite, creatine, on the boundary between the white and gray matter. The observed distribution is consistent with the literature data obtained using magnetic resonance spectroscopy.


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