Session MOF. There are 3 abstracts in this session.

Session: Advances in Technology, time: 4:30 - 4:55 pm

Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis

Joshua Coon
University of Wisconsin-Madison, Madison, WI

Protein glycosylation is a highly important, yet poorly understood protein post-translational modification. Thousands of possible glycan structures and compositions create potential for tremendous site heterogeneity. A lack of suitable analytical methods for large-scale analyses of intact glycopeptides has limited our abilities to both address the degree of heterogeneity across the glycoproteome and to understand how it contributes biologically to complex systems. Here we show that N-glycoproteome site-specific microheterogeneity can be captured via large-scale glycopeptide profiling methods enabled by activated ion electron transfer dissociation (AI-ETD), ultimately characterizing 1,545 N-glycosites (>5,600 unique N-glycopeptides) from mouse brain tissue. Our data reveal that N-glycosylation profiles can differ between subcellular regions and structural domains and that N-glycosite heterogeneity manifests in several different forms, including dramatic differences in glycosites on the same protein. Moreover, we use this large-scale glycoproteomic dataset to develop several visualizations that will prove useful for analyzing intact glycopeptides in future studies.

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Session: Advances in Technology, time: 5:20 - 5:35 pm

Multiplexing meets automation: medium scale-phosphoproteomics assay increases sample throughput and allows for quantification in primary neurons

Katherine DeRuff; Alvaro Sebastian Vaca Jacome; Karen Perez de Arce; Malvina Papanastasiou; James Mullahoo; Deborah Dele-Oni; Steven A. Carr; Jeffrey R. Cottrell; Jacob D. Jaffe
Broad Institute, Cambridge, MA

Phosphoproteomics offers deep insights into cellular signaling and processes, but often requires large amounts of material for standard analyses. In this work, we sought to balance lower phosphoproteomic coverage with increased throughput and reduced sample input requirements, which would allow us to analyze rare cell types and tissues, such as neurons. Expanding upon automation protocols we have previously developed (Abelin et al. 2016), we have now produced a fully-automated, discovery proteomics workflow that leverages Tandem-Mass-Tag labeling and single-shot LC-MS/MS analysis that provides biologically relevant phosphosite information from sample amounts as low as 10 ugd. As a proof-of-principle, we employed mouse and rat cortical neurons and perturbed them with compounds that are known to affect synaptic plasticity for various time points up to 48 hours. The sensitivity of the assay allowed us to analyze 80 samples in triplicates using a total amount of 40 ug per sample. We were able to characterize >5000 phosphopeptides that constitute ~40% of phosphopeptide identifications using traditional phosphoproteomics workflows (Mertins et al. 2018). Mapping of these peptides onto KEGG pathways demonstrates that adequate coverage is achieved to produce a snapshot of important signaling activity at a fraction of the cost and time of traditional phosphoproteomics. The work presented here illustrates the value of the technology to samples with limited amounts.

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Session: Advances in Technology, time: 5:35 - 5:50 pm

Electro-elution chromatography of RNA oligonucleotides: A novel paradigm in RNA analysis by nanoLC-MS/MS

Richard Lauman; Hee Jong Kim; Sam Wein; Kevin Janssen; Benjamin A. Garcia
University of Pennsylvania, Philadelphia, PA

RNA post-transcriptional modifications are ubiquitous and have been associated with splicing events, oxidative stress and in the structure of tRNA and ribosomal RNA. Current nanoLC-MS/MS RNA for post transcriptional modifications methods are limited by the length of the RNA oligo or by ion pairing reagents: losing valuable context in the sequence and from loss of signal due to ion pairing reagent suppression. In this newly designed method, we use a previously known technique that has been so far limited to small molecules to retain and release RNA oligonucleotides selectively by using a novel automated 2D gradient and electro-elution. Using a standard solvent nanoflow LC, a voltage was applied to the porous graphite column over a range to retain and release the RNA molecules. The Electrochemically Modulated Reverse Phase (EMRP) nanoLC-MS/MS method is designed to retain and elute numerous RNA oligomers, solely dependent on their size and charge state. The release of the oligos is dependent on the voltage switch to the column and subsequently the polarity switch of the column. Using synthetic RNA, we determined a limit of detection (LODs) in the range of 100-200 attomole/uL, dependent on sequence of RNA. This detection limit is well within the possible concentrations in cells and even serum. MS1 and MS2 data has been processed with the use of a novel program developed in house (NucleicAcidSearchEngine, NASE), which can discern the higher charge states and determine sequences of the RNA oligonucleotides with an applied FDR. Combined with this computational software, we now have complete platform to detect smaller sequences of RNA, such as microRNAs or RNA digested with nuclease from any cellular source, removing much of the issues surrounding PCR amplification.

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