{'fraction_non_alphanumeric': 0.04977865597628864, 'fraction_numerical': 0.027112267968764845, 'mean_word_length': 4.985897873308313, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
C-terminal methylation of truncated neuropeptides: An enzyme-assisted extraction artifact involving methanol

Neuropeptides are the largest class of signaling molecules used by nervous systems. Today, neuropeptide discovery commonly involves chemical extraction from a tissue source followed by mass spectrometric characterization. Ideally, the extraction procedure accurately preserves the sequence and any inherent modifications of the native peptides. Here, we present data showing that this is not always true. Specifically, we present evidence showing that, in the lobster Homarus americanus, the orcokinin family members, NFDEIDRSGFG-OMe and SSEDMDRLGFG-OMe, are non-native peptides generated from full-length orcokinin precursors as the result of a highly selective peptide modification (peptide truncation with C-terminal methylation) that occurs during extraction. These peptides were observed by MALDI-FTMS and LC-Q-TOFMS analyses when eyestalk ganglia were extracted in a methanolic solvent, but not when tissues were dissected, co-crystallized with matrix, and analyzed directly with methanol excluded from the sample preparation. The identity of NFDEIDRSGFG-OMe was established using MALDI-FTMS/SORI-CID, LC-Q-TOFMS/MS, and comparison with a peptide standard. Extraction substituting deuterated methanol for methanol confirmed that the latter is the source of the C-terminal methyl group, and MS/MS confirmed the C-terminal localization of the added CD3. Surprisingly, NFDEIDRSGFG-OMe is not produced via a chemical acid-catalyzed esterification. Instead, the methylated peptide appears to result from proteolytic truncation in the presence of methanol, as evidenced by a reduction in conversion with the addition of a protease-inhibitor cocktail; heat effectively eliminated the conversion. This unusual and highly specific extraction-derived peptide conversion exemplifies the need to consider both chemical and biochemical processes that may modify the structure of endogenous neuropeptides.


Introduction
Peptides are the largest class of signaling molecules used by nervous systems to modulate physiology and behavior. Members of this class of signaling agents can function as locally released neuromodulators and/or as circulating hormones. Peptides are initially synthesized as larger prepro-hormones, which undergo at least one cleavage, and often extensive post-translational modification, prior to assuming their final bioactive conformations [7].
Crustaceans, particularly members of the Decapoda, have a long history in peptide research [7]. In these animals, mass spectrometry (MS) has played a major role in peptide discovery [20,28]. The MS-based identification of neuropeptides from crustaceans has frequently relied upon matrix assisted laser desorption/ionization (MALDI)-based analysis of small tissue samples removed from an individual animal by microdissection techniques (direct tissue analysis). Alternatively, peptides can be extracted from single tissues or tissues pooled from many individuals prior to MALDI or electrospray ionization (ESI). Regardless of method, the identification of novel neuropeptides relies upon the assumption that the tissue isolation/preparation and/or extraction procedures used accurately preserve the sequence and any inherent modifications of the native peptides.
One group of crustacean peptides that has been the subject of extensive MS investigations is the orcokinin family, members of which are typified by an overall length of 13 amino acids and the structure NFDEIDRXXXGFX, where X represents a variable residue [7]. First described from the crayfish Orconectes limosus [41], members of this peptide family have subsequently been identified from many crustacean species (summarized in [7]), with many members identified by MS-based analysis. In most crustaceans, multiple orcokinins are present, all derived from a common prepro-hormone, which is also the source of a variety of peptides in addition to the orcokinins. For example, in the American lobster Homarus americanus, the orcokinin precursor protein contains the orcokinins NFDEIDRSGFGFN (3 copies), NFDEIDRSGFGFH (2 copies) and NFDEIDRSGFGFV (2 copies), as well as one copy each of FDAFTTGFGHN (commonly referred to as ocomyotropin), SSEDMDRLGFGFN (an orcokinin-like peptide), GPIKVRFLSAIFIPIAAPARSSPQQDAAAGYTDGAPV, GDYDVYPE, VYG-PRDIANLY and SAE [10].
In recent MS-based analyses of H. americanus neural tissues, each of the full-length orcokinins was detected, as were FDAFTTGFGHN, SSEDMDRLGFGFN, GDYDVYPE, and VYGPRDIANLY [10]. In addition, a number of truncated orcokinin and orcokininlike peptides were characterized in this study [10] and in studies by other researchers [4,6,10,27,40]. Surprisingly, in studies conducted by Li and colleagues, several truncated orcokinins seemingly not derived from the known full-length peptides, i.e. NFDEIDRS-GFA and SSEDMDRLGFA, were characterized [30]; NFDEIDRSGFA has also been sequenced via MS-analysis from the crab Cancer borealis [21], where it too does not correspond to any of the known full-length orcokinin isoforms. Given this discrepancy, we became interested in determining the origin of NFDEIDRSGFA and SSEDMDRLGFA in the lobster. In the data that follow, we present evidence showing that extraction with acidified methanol yields the C-terminally methylated peptides NFDEIDRSGFG-OMe and SSEDMDRLGFG-OMe, and find no evidence to support the detection of NFDEIDRSGFA and SSEDMDRLGFA in H. americanus, suggesting that these peptides have been mis-identified. Using high resolution MALDI-Fourier transform mass spectrometry measurements, sustained off-resonance irradiation collision-induced dissociation (SORI-CID), high performance liquid chromatography-Chip nano-electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC Chip-nanoESI Q-TOF MS), and isotopic labeling, we also show that methylation at the C-terminus of these truncated peptides arises as the result of a highly selective peptide modification during neuropeptide extraction from certain crustacean tissue samples in the presence of methanol, a solvent commonly used for peptide extraction. Furthermore, we show that this modification is not a simple chemical artifact, but rather is likely an enzymatically mediated process involving methanol. Taken collectively, the data presented in this study demonstrate the need to consider that unexpected neuropeptide modifications may occur in the process of neuropeptide extraction from tissue samples, giving rise to artifactual isoforms that can be misconstrued as naturally occurring, native isoforms.

Animals and tissue collection
American lobsters, H. americanus, were purchased from local suppliers (Brunswick and Harpswell, ME, USA) and maintained in LG), the medulla externa (ME), the medulla interna (MI), the medulla terminalis (MT), the X-organ (XO), and the sinus gland (SG). The XO is a collection of neurosecretory cells located within the MT, which produce hormones that are released to the SG. The XO and SG are linked by the sinus gland tract (sgt). The optic nerve (optn) connects the eyestalk to the supraesophageal ganglion (SoG). aerated seawater tanks at 8-10 • C. Prior to dissection, animals were anaesthetized by packing in ice for approximately 30 min. After icing, the eyestalk ganglia were isolated via manual microdissection in chilled (8-10 • C) physiological saline. For some experiments, eyestalk ganglia were divided in a regional specific manner to allow for analysis of the individual regions of this system, i.e. the medulla terminalis (MT), which includes the X-organ (XO), the medulla interna (MI), the medulla externa (ME), the lamina ganglionaris (LG), and the sinus gland (SG), a neuroendocrine organ derived from the XO somata (Fig. 1). Techniques used for the isolation of entire stomatogastric ganglion (STG) and commissural ganglion (CoG), or small pieces of the supraesophageal ganglion (brain), or pericardial organ (PO) have been described [10,11].

Sample preparation for direct tissue MALDI-FTMS analysis
For direct tissue MALDI-FTMS, dissected tissues were rinsed sequentially in two 12 L droplets of 0.75 M d-fructose solution (reagent grade; Fisher Scientific, Pittsburgh, PA, USA) and placed on a face of a ten-faceted probe tip, minimizing co-transfers of solution. The tissue was then sliced 10-20 times with a 0.2 mm minuten pin, gathered together, and covered with a 0.5 L droplet of 1.0 M 2,5-dihydroxybenzoic acid [DHB; Sigma-Aldrich (sublimed prior to use)] prepared in 1:1 acetonitrile:water containing 2% phosphoric acid.

Extraction with methanol:water:acetic acid
For most extractions in acidified methanol, the extraction solvent was 30% deionized water, 65% methanol (CH 3 OH; HPLC-grade; Fisherbrand), and 5% glacial acetic acid (CH 3 CO 2 H; reagent grade; Sigma-Aldrich, ≥99%), as a %[v/v] mixture. A single eyestalk ganglion was rinsed sequentially in two 12 L droplets of 0.75 M d-fructose solution, placed in a 0.6 mL low retention centrifuge tube (Fisherbrand) with 50 L of extraction solvent (smaller volumes were used when smaller tissues were analyzed), and then homogenized by one of the following methods. In early work, the tissue was repeatedly sliced with spring scissors; in most of the work reported in this study, tissues were ground by inserting a longer, smaller diameter polypropylene tube (0.25 mL; Fisherbrand) into the 0.60 mL tube and repeatedly twisting the tube for homogenization. In some experiments, deuterated methanol (CD 3 OD; 99.8% deuterated; Cambridge Isotope Laboratories, Andover, MA, USA) was substituted for the standard CH 3 OH in the extraction buffer (the same solvent composition was used). After tissue homogenization, the sample was sonicated for 2-5 min and centrifuged at 15k rpm for 5-15 min. The supernatant was removed from the sample and placed in another 0.6 mL tube. In early experiments, samples were delipidated prior to analysis. For delipidation, 25 L of nanopure water was added to the supernatant along with 25 L chloroform (NMR-grade 13 CDCl 3 ; Cambridge Isotope Laboratories) in order to remove lipids from the aqueous solution. The two layers were sonicated for 2 min and centrifuged for 10 min. The bottom organic layer was removed. Chloroform was added and the extraction was repeated two more times, but with a 5-min centrifugation.
The aqueous layer was either stored at −20 • C or concentrated to dryness in a SpeedVac vacuum concentrator (UVS400 Universal Vacuum System, Thermo Electron Corporation) at 36 • C. Once dried, the extract was reconstituted to a total volume of 50 L in 1:1 ACN:H 2 O in preparation for analysis by MALDI-FTMS or HPLC Chip-nanoESI Q-TOF MS. For some samples analyzed by MALDI-FTMS, the extracts, reconstituted in 0.1% TFA water, were desalted using C18 ZipTip pipette tips (Millipore, Billerica, MA, USA). For MALDI-FTMS analysis of extracts, 0.5 L of the extract was mixed with 0.5 L of DHB matrix on one face of the MALDI probe and the extract-matrix mixture was allowed to co-crystallize.

Extraction with acetone:water:hydrochloric acid or saturated DHB
For extractions in acidified acetone (85% acetone [Sigma-Aldrich, ≥99%], 13% deionized water, and 2% HCl [reagent grade; Fisherbrand], as a %[v/v] mixture, a single eyestalk ganglion was rinsed sequentially in two 12 L droplets of 0.75 M d-fructose solution and placed in a 0.6 mL low retention centrifuge tube (Fisherbrand) with 50 L of extraction solvent. The tissue was ground by inserting a longer, smaller diameter polypropylene tube (0.25 mL; Fisherbrand) into the 0.60 mL tube and repeatedly twisting the tube for homogenization. After tissue homogenization, the sample was sonicated for 2-5 min and centrifuged at 15k rpm for 5-15 min. The supernatant was removed from the sample and dried prior to being reconstituted in 1:1 ACN:H 2 O in preparation for analysis by MALDI-FTMS.
For extraction in saturated DHB, the extraction protocol described above was followed, using 50 L of a freshly prepared, saturated solution of DHB in deionized water as the extraction solvent.

Extractions with protease inhibitors
Paired eyestalk ganglia were dissected from individual lobsters, with the ganglion from one eyestalk used as a control and the ganglion from the second used as a test to determine if a protease inhibitor cocktail, included in the extraction protocol, reduces or eliminates the C-terminal methylation reaction. The protease inhibitor cocktail was prepared by dissolving one tablet (complete, Mini; Roche Applied Science, Indianapolis, IN, USA) in 1.5 mL deionized water to prepare a stock solution, which was further diluted 1:7 with deionized water. In initial experiments, the control eyestalk tissue was homogenized in normal extraction solvent (65:30:5, methanol:water:acetic acid), while the test eyestalk tissue was homogenized in extraction solvent in which water had been replaced with protease inhibitor cocktail solution. After homogenization, the tissues were sonicated for 5 min, centrifuged for 15 min, and the supernatant was removed from the tissue pellet.
In later experiments, the control tissue was first homogenized and sonicated in 30 L of nanograde water; the test tissue was homogenized and sonicated in 30 L protease inhibitor cocktail solution. Then, 65 L of methanol and 5 L of glacial acetic acid were added to each tube. The samples were resonicated and centrifuged; the supernatant was then removed from the tissue pellet. Most samples were dried and subjected to ZipTip purification prior to analysis.

Extractions with heat
Paired eyestalk ganglia were dissected from individual lobsters, with the ganglion from one eyestalk used as a control and the ganglion from the second used to test the effect of submerging the tissue in boiling water prior to homogenization. Each tissue was placed in 50 L of normal extraction solvent. The control tissue sample sat at room temperature for 5 min; the test tissue sample was placed in a boiling water bath for 5 min. The two samples were then homogenized, sonicated, centrifuged and the supernatant was removed from the tissue pellet.

Liquid nitrogen freezing of tissues
Prior to the standard tissue extraction procedure detailed above, the ganglion from one eyestalk was immediately placed in a beaker of liquid nitrogen with forceps for 15 s in order to freeze the tissue. The tissue was then placed in a 0.6 mL microcentrifuge tube and homogenized by grinding with a smaller centrifuge tube. Then 30 L of extraction solvent was added to each tissue sample and the tissues were then extracted as described above.

Standards with extraction solvent
To determine whether methyl esterification occurs with synthetic standards, 2 L of 10 −3 M [Asn 13 ]-orcokinin or Orc [1][2][3][4][5][6][7][8][9][10][11] (NFDEIDRSGFGFN or NFDEIDRSGFG, respectively; GenScript Corporation, Scotch Plain, NJ, USA) was mixed with 30 L of either CH 3 OH:water:acetic acid (65:30:5), CD 3 OD:water:acetic acid (65:30:5), or nanopure water. The solutions sat at room temperature for 23 h before they were dried, reconstituted with 25 L 1:1 ACN:water, and analyzed by MALDI-FTMS.  30:5)]. The first tube contained only the solvent and standard; to the second tube, one eyestalk ganglion was added and homogenized. Both samples were sonicated for 5 min and centrifuged for 15 min; the solvent fraction was then analyzed by MALDI-FTMS. The solvent in the tissue-containing samples was analyzed without separating the supernatant from the tissue pellet. An additional 1 nmol of the [Ala 13 ]-orcokinin standard was added to the tissue/extraction solvent mixture, and the sample was resonicated and centrifuged and analyzed before and after sitting at room temperature overnight.

Tryptic digestion of NFDEIDRAAFGFA in methanolic buffer
To determine whether C-terminal methylation can compete with hydrolysis by trypsin, 1 nmol of NFDEIDRAAFGFA was mixed with 0.09 nmol of bovine trypsin (Sigma-Aldrich) in 25 mM, pH = 4.0, citrate buffer prepared with water or 25% methanol. The digestion proceeded at room temperature for 1-4 days with analysis by HPLC Chip-nanoESI Q-TOF MS.

MALDI-FTMS
Most samples were analyzed using a HiResMALDI Fourier transform mass spectrometer (Varian, Lake Forest, CA, USA) with a Cryomagnetics (Oak Ridge, TN, USA) 4.7 Tesla actively shielded superconducting magnet. Ions were generated using a pulsed nitrogen laser (337 nm) and were transported from the external ion source to the closed cylindrical ICR cell using a quadrupole ion guide. The ion guide radio frequency potential and trapping delay time were optimized to transmit and trap ions of a selected mass range (optimized for m/z 1500 for the results presented here). A pulse of argon was introduced to the vacuum system during trapping to elevate the system pressure transiently for collisional cooling. All spectra were measured using ion accumulation techniques, where ions from 7 to 30 successive laser shots were accumulated in the cell. A delay of 5-10 s preceded ion detection, which occurred with analyzer pressures of 1-2 × 10 −10 Torr. Transients (4 or 10 s) from direct tissue spectra were apodized using a Blackman function and zero-filled prior to fast Fourier transformation; SORI-CID spectra (see below) were processed without apodization.
Mass spectra were calibrated using the m/z values for two previously identified peptides [2]: APSGFLGMRamide (C. borealis tachykinin-related peptide I [CabTRP I]; m/z = 934.4927) and VYRKPPFNGSIFamide (Val 1 -SIFamide; m/z = 1423.7845). Peaks for these two peptides were present in the spectra and served as internal calibrants.
For SORI-CID experiments, argon was used as the collision gas, the frequency offset was set to −1.8% of the reduced cyclotron frequency and the voltage amplitude was in the range of 6-8.5 V bp . SORI-CID spectra were calibrated externally, with a one-point adjustment based upon a [MH−NH 3 ] + fragment mass. The standards NFDEIDRSGFGA and NFDEIDRSGFG-OMe were characterized by SORI-CID.

HPLC Chip-nanoESI-Q-TOF MS
Mass spectrometric analysis was performed using a 6530 quadrupole time-of-flight (Q-TOF) mass analyzer (Agilent Technologies, Santa Clara, CA). Mass spectra (MS and MS/MS) were collected in positive ion mode; the ionization voltage ranged from 1850 to 1950 V and the ion source temperature was held at 350 • C. Spectra were internally calibrated using methyl stearate (C 17 H 35 CO 2 CH 3 ) and hexakis(1H, 1H, 4Hhexafluorobutyloxy)phosphazine (HP-1221; C 24 H 18 O 6 N 3 P 3 F 36 ), continuously infused and detected as [M+H] + . CID-MS/MS experiments were executed with precursor ions subjected to CID using nitrogen as the target gas with the collision energy set to 26 V. Chromatographic separation and nano-electrospray ionization (ESI) were performed using a 1260 Chip Cube System (Agilent Technologies) using a ProtID-chip with a 40 nL enrichment column and a 43 or 150 mm × 75 m analytical column (Agilent Technologies). The enrichment and analytical columns were packed with 300Å, 5 m particles with C18 stationary phase. The mobile phases were 0.1% formic acid/H 2 O (A) and 0.1% formic acid/acetonitrile (B). Samples

Data analysis and figure production
Calibrated mass spectral peak lists were generated using the Omega 8.0 software (IonSpec, Lake Forest, CA, USA). MALDI-FTMS figures were generated using the Boston University Data Analysis software (B.U.D.A.; provided by Dr. Peter O'Connor, University of Warwick, Department of Chemistry, Coventry, England) to produce graphics that were imported into CorelDRAW X4 for final figure production. HPLC Chip-nanoESI Q-TOF MS figures were generated by exporting Mass Hunter (Agilent) chromatograms or mass spectra as metafiles and importing these graphics into CorelDRAW X4 for final figure production.

Results
The paired eyestalks of the lobster, H. americanus, and other crustaceans are composed of several distinct regions (shown schematically in Fig. 1) that contain unique complements of neuropeptides [18]. Only the SG of H. americanus has been characterized by mass spectrometry [6,8,10,15,30], and this tissue has been found to be a rich source of crustacean neuropeptides. This study was originally initiated in an attempt to more fully characterize the complement of neuropeptides present in different regions of H. americanus optic (eyestalk) ganglia, using MALDI-FTMS and the direct analysis of tissue samples, complemented by the analysis of tissue extracts. For this study, the analysis of tissue extracts was needed to characterize tissues that were too large to be fully characterized directly. We also planned to extract and analyze entire optic ganglia to obtain a full measure of the neuropeptide components. For the extraction of neuropeptides from tissue samples, we used an approach applied in previous studies in our lab, which involved microdissection of the desired tissue, tissue homogenization in a methanolic solvent mixture (65:30:5, methanol:water:glacial acetic acid for the work reported here), followed by sonication and centrifugation prior to MALDI-FTMS analysis. In early experiments, samples were delipidated with chloroform. This step, which did not impact our results, was eliminated for most work reported here.
An important aspect of the work described below is an appreciation of the nature of the neuropeptide signals produced on our MALDI-FTMS instrument, which is particularly relevant to the identification of orcokinin family peptides. As described in previous publications [10,43], orcokinin family peptides produce a unique mass spectral signature, characterized by the fragment ions summarized in Fig. 2B, when analyzed by our vacuum UV-MALDI-FTMS instrument. The mass spectral signature is a consequence of the orcokinin amino acid sequence, which incorporates a basic, proton sequestering, arginine (R) residue and two acidic aspartate (D) residues. When a peptide sequence containing this amino acid combination is coupled with the energy imparted by the vacuum UV-MALDI ionization and the long trapping times required for FTMS analysis (>10 s), singly protonated orcokinin family peptides undergo so-called "Asp-Xxx cleavages" [47], which result in the production of characteristic C-terminal (y-type) fragments (see Fig. 2B). Our identification of orcokinin family peptides by MALDI-FTMS relies on the detection of both the [M+H] + ion and the . All spectra were measured using DHB as the matrix and conditions optimized for m/z 1500. Spectra were calibrated using known peptide peaks [42], including APSGFLGMRamide (CabTRP) and VYRKPPFNGSIFamide (Val 1 -SIFamide) at m/z 934.4927 and m/z 1423.7845, respectively. The peaks at m/z 1270.57, 1253.54, 894.43, 876.42, and 537.28 (labeled with filled circles) in (C) and (D) were attributed to an orcokinin family peptide that was not detected in directly analyzed tissues, such as (A); peaks for Orc [1][2][3][4][5][6][7][8][9][10][11]  observation of characteristic y-type ions resulting from Asp-Xxx cleavages.

MALDI-FTMS analysis of eyestalk tissue extracts shows an orcokinin family peptide not predicted by genomics
When we analyzed small pieces of eyestalk ganglion tissues directly by MALDI-FTMS, we detected neuropeptide peak profiles that reflected differential distributions of neuropeptides in localized regions of the eyestalk ganglia. For example, the peptides CabTRP I (APSGFLGMRamide at m/z 934.49) and Val 1 -SIF (VYRKPPFNGSIFamide at m/z 1423.78) were detected in tissues from the LG, XO/MT, MI, and ME but not in the SG. Orcokinin family peptides were detected in many tissues, including the XO/MT, MI, ME, and SG. A representative spectrum from a small piece of XO/MT tissue is shown in Fig. 3A.
In contrast with previous studies [10], where we found good agreement between single tissues analyzed directly and by single tissue extraction, our analysis of extracts of tissues from the aforementioned regions of the eyestalk ganglion revealed the presence of a new peptide that had not been detected by direct tissue MALDI-FTMS. For example, Fig. 3C shows the spectrum observed when a small piece of XO/MT tissue was removed by microdissection techniques, placed in extraction solvent, homogenized, sonicated, and centrifuged. While we continue to detect peaks for CabTRP I and Val 1 -SIF, an abundant signal at m/z 1270.57 was observed, which was detected in combination with additional peaks showing the characteristic orcokinin family pattern. The full collection of peaks appeared at m/z 1270.57, 1253.54, 894.43, 876.42, and 537.28; these peaks were assigned to [M+H] + , [MH−NH 3 ] + , y n+4 , y o n+4 , and y n+1 . Unexpectedly, these masses did not correspond to any orcokinin family members predicted from genomic information for H. americanus [10], nor did they correspond to masses expected from conventional post-translational modifications, including the truncation of full-length orcokinin family peptides. Instead, exact mass measurements (m/z 1270.5692, measured) were consistent with the orcokinin sequence, NFDEIDRSGFA (Orc[Ala 11 ]; m/z 1270.5699, predicted). This peptide has been detected in other studies [4,16,19,30,31] with the first characterization, from the crab, C. borealis, reported in a study by Huybrechts et al. [21]; later reports include the detection of Orc[Ala 11 ] in nervous system tissues and neuroendocrine organs from H. americanus [30]. While C-terminally truncated versions of full-length H. americanus orcokinin-family peptides, including Orc [1][2][3][4][5][6][7][8][9][10][11][12] and Orc [1][2][3][4][5][6][7][8][9][10][11] ( Fig. 2A), detected in XO/MT extract (Fig. 3C) and direct tissue ( Fig. 3A and B) spectrum, have been also been reported by our laboratory [10] and by other researchers [4,6,10,27,40], the alaninecontaining peptide sequence is unusual because an alanine residue at this position is not known for any full-length orcokinins detected mass spectrometrically or predicted from genomic information.
When we analyzed the extract from an entire eyestalk ganglion, we again detected peaks for the m/z 1270.57, putative Orc[Ala 11 ], peptide (see Fig. 3D). To ensure that the detection of this peptide was not the result of a mutation specific to the individual animal analyzed, localized tissue samples and entire eyestalk ganglia from additional individuals (n > 30) were extracted. Although the abundance of the m/z 1270.57 and other putative Orc[Ala 11 ]-derived peaks varied relative to that of other detected peptides, signals for this peptide were consistently observed, except in extracted sinus gland samples, where these signals were weak or missing.

MALDI-FTMS with SORI-CID supports the identification of NFDEIDRSGFG-OMe, not NFDEIDRSGFA
To further characterize the amino acid sequence of the peptide appearing at m/z 1270.57, we subjected the peak to analysis by SORI-CID, the form of MS/MS used on our FTMS instrument. Isolation of the [M+H] + at m/z 1270.57 from an eyestalk ganglion extract followed by SORI-CID yielded a spectrum showing an abundant peak at m/z 1253.54 (loss of NH 3 ) and the production of y-type sequence ions, including the Asp-Xxx cleavage products at y 8 , y o 8 , and y 5 (m/z 894.43, 876.42, and 537.28, respectively; see Fig. 4A). This experiment provided support for our assignment of the m/z 1270.57 peak as an ionized orcokinin family peptide and, furthermore, supported our attribution of the m/z 1253.54, 894.43, 876.42, and 537.28 peaks in the MALDI-FT mass spectrum of tissue extracts to this gas-phase precursor. However, the SORI-CID mass spectrum did not provide sufficient information to establish the full amino acid sequence.
In previous studies [43], we have shown that the variable Cterminal sequence of orcokinin-family peptides can be established by using the mass spectrometric isolation and dissociation of the Each m/z 1270.57 peak was dissociated following ion isolation. Argon was used as the collision gas with an excitation amplitude of 6.5 V bp . The y-type fragment ions are identified using the nomenclature established by Roepstorff [36]. y n+1 fragment by SORI-CID. The y n+1 fragment, produced via Asp-Xxx cleavage, contains the arginine (R) residue at the N-terminus and yields b-type sequence ions, which retain the N-terminal, arginine-containing, end of the peptide sequence. When the y 5 peak at m/z 537 was isolated and subjected to SORI-CID interrogation, we measured peaks, including b 1 , b 2 -NH 3 , b 3 , and b 4 at m/z 157.11, 227.11, 301.16, and 448.23, respectively, that are consistent with the sequence RSGF (Fig. 5A). Other peaks in the spectrum (m/z 472. 23  The SORI-CID mass spectrum of the y5 peak at m/z 537.28, which was produced from an Orc[Ala 11 ] standard. The spectrum shows the [b4+H2O] + ion at m/z 466.24, which is generated by fragmentation of peptides with a free acid at the C-terminus. (C) The SORI-CID mass spectrum of the [M+H] + , m/z 537.28, y5 peak produced from an Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe standard. The [b4+H2O] + ion at m/z 466.24 is absent because methylation of the C-terminus blocks the rearrangement [45] required for ion formation. Each m/z 537.28 peak was dissociated following ion isolation. Argon was used as the collision gas with an excitation amplitude of 6.5 V bp . The y-and b-type fragment ions are identified using the nomenclature established by Roepstorff [36].
To more conclusively establish if the m/z 1270.57 peptide is, in fact, Orc[Ala 11 ], we measured SORI-CID mass spectra for a synthetic form of the peptide. As shown in Fig. 4B, SORI-CID of the m/z 1270.57, [M+H] + , peak yields a spectrum that closely resembles that of the eyestalk extract-derived peptide; however, we note that the intensity of the y 8 peak (m/z 894.43) for the standard (Fig. 4B) is consistently lower than that observed for the putative Orc[Ala 11 ] peptide (Fig. 4A). While this mass spectral difference was reproducible, the fact that the mass spectrum is dominated by Asp-Xxx cleavage ions (y 8 , y o 8 , and y 5 ) limited our ability to carry out a more detailed comparison of structural features. In contrast, SORI-CID of the y 5 peak at m/z 537.28 proved to be more revealing. While similar ions and ion intensities were detected in the lower m/z range of the spectrum, more significant differences in ion intensities and ion identity were observed at higher m/z values (Fig. 5B). Most notably, the SORI-CID spectrum of the Orc[Ala 11 ]-derived y 5 peak ( Fig. 5B) shows an abundant [b 4 +H 2 O] + product ion at m/z 466.24, which was not detected in the spectrum of the eyestalk extractderived peptide (Fig. 5A). Production of the [b 4 +H 2 O] + ion from the Orc[Ala 11 ]-derived y 5 ion is congruent with predicted fragmentation behavior, based upon studies of other orcokinin peptides (described above). The fact that this peak is not detected in the spectrum of putative Orc[Ala 11 ] provides defining evidence that our eyestalk extract-derived peptide is not Orc[Ala 11 ].
Furthermore, when structural elements that would block formation of the [b 4 +H 2 O] + ion are considered, we are able to propose a sequence for the eyestalk-derived m/z 1270.56 orcokinin peptide. Specifically, the mechanism responsible for the production of [b n−1 +H 2 O] + product ions has been investigated, and it is known that ion formation involves a rearrangement at the C-terminus that requires a free C-terminal carboxyl group [45]. This rearrangement is prevented when the C-terminus is blocked by amidation or esterification. Based upon this information, we hypothesized that the m/z 1270.57, eyestalk extract-derived peptide was not Orc[Ala 11 ], but was, instead, NFDEIDRSGFG-OMe (also m/z 1270.57), a C-terminally truncated orcokinin that has been specifically methylated at the C-terminus. To test this hypothesis, we carried out the SORI-CID characterization of synthetic Orc  While LC retention times proved to be diagnostic for distinguishing Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe and Orc[Ala 11 ], CID experiments carried out by HPLC Chip-nanoESI Q-TOF MS yielded MS/MS spectra for the two standard and the eyestalk-extract peptide that were virtually indistinguishable (see Fig. 7A, E, and G). In contrast with SORI-CID mass spectra of these compounds, where dissociation of the m/z 1270.56, [M+H] + precursor ions (Fig. 4A-C) provided very limited sequence information as a consequence of proton localization by the charge-sequestering arginine residue, dissociation of the m/z 635.79, [M+2H] 2+ , precursor ions on the Q-TOF instrument yielded MS/MS spectra that provided excellent sequence coverage through the formation of y-and b-type ions (see Fig. 7A, E, and G); however, the MS/MS spectra still precluded structural differentiation because product ion masses were identical (Ala and G-OMe are isobaric) and the structurally similar residues did not influence relative ion intensities in ways that were useful for distinguishing the two peptide sequences.

Methanol in the extraction solvent is the source of added methyl group
Because our extraction solvent contains 65% methanol, the methanolic solvent was hypothesized to play a role in the production of Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe. To determine if this was the case, we carried out tissue extractions using CD 3 OD, instead of CH 3 OH, in the extraction solvent. To eliminate variations that may result when comparing tissues extracted from different individuals, the paired eyestalk tissues were removed from one lobster. One eyestalk ganglion was placed in extraction solvent containing CH 3 OH and the other in extraction solvent containing CD 3 OD. The samples were homogenized, sonicated, and centrifuged, and the supernatant was separated from the tissue pellet. The samples were dried and reconstituted for analysis by MALDI-FTMS. The MALDI-FT MALDI-FT mass spectra and CID experiments carried out on the Q-TOF also support our localization of the added methyl group at the C-terminus of the peptide. This localization is supported by the 3 Da mass shifts for the y 8 , y 0 8 , and y 5 ion in the MALDI-FT mass spectra of eyestalks extracted with CD 3 OD (Fig. 8), which localizes the added methyl group to the C-terminal sequence, and by the 3 Da mass shift for y-type (but not b-type) ions produced in the Q-TOF MS/MS analysis ( Fig. 7C and D). The most diagnostic fragment is the y 1 peak, which undergoes a 3 Da mass shift (from m/z = 90.06 to 93.07) for the CD 3 -labeled peptide (Fig. 7D). This fragment ion definitively localized the methyl addition to the C-terminus. These results document the incorporation of one CD 3 group for Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe at the C-terminus and demonstrate that the methanolic extraction solvent is the source of the added methyl group.

Methylation is a biochemical, not chemical, extraction artifact
Acid-catalyzed esterifications have been recognized as the source of exogenous protein methylations that occur when methanol and acids are used, for example, in destaining SDS-PAGE gels [5,17,24,48]. In an early study by Haebel et al. [17], five test peptides were incubated in methanolic trichloroacetic acid (TCA) solutions (12.5:50:37.5, methanol:TCA:water) for 1-24 h to determine the propensity for methylation at different amino acid residues. The authors concluded that glutamate (E) undergoes the most rapid acid-catalyzed esterification, the C-terminus reacts with a rate that is lower by a factor of 2-6, and other groups (D, Q) are less reactive by at least a factor of 10 [17]. When acetic acid replaced TCA, methylation was not observed [17].
A direct chemical modification appeared to be unlikely as an explanation for the production of Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe under our conditions, based upon the following observations and considering the work by Haebel et al. [17]. First, our data clearly show that methylation occurs, with 100% specificity, at the C-terminus. It seems unlikely that an acid-catalyzed chemical conversion could account for this highly specific methylation, which leaves the remaining two aspartate and the single glutamate residues unmodified. Second, we find no evidence for methylation of any full-length orcokinin family peptides. Third, we find that significantly lower levels of Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe are found in extracts from the SG (a neuropeptide storage site) compared with extracts of whole eyestalk ganglia or small pieces of eyestalk tissue, such as the XO/MT, where enzymes important for the synthesis and processing of neuropeptide prohormones are expected to be co-localized. Based upon these observations, we hypothesized that methylation must involve processing components endogenous to the eyestalk tissues.

Enzyme inhibitors reduce Orc[1-11]-OMe formation
To determine if enzymes play a role in promoting the formation of Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe during extraction of eyestalk tissues, we attempted to reduce methylation by inhibiting enzymatic activity using a commercial protease inhibitor cocktail that contains a mixture of inhibitors designed to protect against a broad range of proteases. To include the protease inhibitor in our extraction protocol, we used two different approaches. The first approach involved including the aqueous inhibitor solution in place of water in our extraction solution. The second approach involved homogenizing the tissue in either the aqueous inhibitor solution or water (as a control), followed by the addition of acidified methanol to bring the solvent to the percentages that have been used in previous experiments. Experiments were carried out with paired eyestalk ganglia to directly compare the efficacy of the inhibitor treatment.

Heat treatment significantly reduces Orc[1-11]-OMe formation; treatment with liquid N 2 does not
Heat has been used an effective means to reduce proteolytic degradation of proteins when processing vertebrate tissue samples [9,12,13,44]. Working from the hypothesis that an enzyme plays a role in promoting the formation of Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe during extraction of eyestalk tissues, we attempted to deactivate enzymatic components with heat. To test this approach we removed the paired eyestalk ganglia from one lobster. The ganglion from a single eyestalk was placed in a microcentrifuge tube with 50 L of extraction solvent and the tightly capped tube was placed in a boiling water bath for 5 min. Concurrently, the ganglia from the second eyestalk of the same lobster were placed in extraction solvent and left at room temperature for 5 min. Both eyestalk tissue samples were then homogenized, sonicated, and centrifuged prior to MALDI-FTMS analysis.
We also tried freezing eyestalk ganglion tissues in liquid nitrogen before homogenizing and adding extraction solvent, but found that this treatment did not measurably reduce production of Orc[1-11]-OMe.
orcokinin. Collectively, these experiments provide evidence that the percentage of water, as well as solution pH, play a role in the putative enzymatic conversion of orcokinin family peptides to hydrolyzed or methylated, truncated forms.

Trypsin-catalyzed C-terminal methylation at low pH is demonstrated for NFDEIDRAAFGFA
To further test the hypothesis that an enzyme is responsible for the formation of Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe, and that methanolysis can compete with proteolysis at acidic pH values that are not optimal for enzymatic activity [3], we carried out the proteolysis of NFDEI-DRAAFGFA, a synthetic peptide, with and without 25% methanol in a pH = 4, 25 mM citric acid buffer. We used trypsin as a representative serine protease that should permit methanol to act as a competing nucleophile in the proteolysis reaction [2,38]. Following reaction, the products were analyzed by Chip-nanoESI Q-TOF MS. In the control experiment (no methanol present), trypsin cleaved the peptide C-terminal to the arginine residue to produce two hydrolysis products, NFDEIDR and AAFGFA (see Fig. 13A). In the presence of methanol, C-terminal methylation was observed through the formation of NFDEIDR-OMe (see Fig. 13B). No other methylated products were observed.

An additional truncated, methylated peptide, SSEDMDRLGFG-OMe, is identified
To determine if additional methylated peptides were present in eyestalk extracts, we compared spectra of eyestalks extracted with CH 3 OH with those extracted with CD 3 OD and searched for peptides that showed the predicted shift of 3 Da. This analysis results in the identification of the peptide SSEDMDRLGFG-OMe, which showed peaks at m/z 1227.53, 809.40, and 563.33 ([M+H] + , y 7 , and y 5 , respectively). Exact mass measurements (1227.5298, measured; 1227.5310, predicted) supported this assignment. This peptide is presumably derived from the full-length precursor SSED-MDRLGFGFN. Fig. 13. Chromatograms from the HPLC Chip-nanoESI Q-TOF MS analysis of a tryptic digestion of synthetic peptide NFDEIDRAAFGFA in (A) pH = 4 citrate buffer and (B) 25% CH3OH, pH = 4 citrate buffer, demonstrating that, under appropriate conditions, a serine protease can accommodate methanol as a competing nucleophile and generate a product methylated at the C-terminus. Peaks 1 (NFDEIDR) and 3 (AAFGFA) result from tryptic cleavage C-terminal to the arginine residue. Peak 4 is the undigested peptide. Peak 2 results from C-terminal methylation when methanol was present in the solvent, forming NFDEIDR-OCH3. Samples were digested for 3 days at room temperature; starting peptide concentration was 2 × 10 −6 M.
3.15. Direct-tissue analysis by MALDI-FTMS reveals no peaks for Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe/Orc[Ala 11 ] in H. americanus neuroendocrine organs and tissues When we initially embarked upon our study of localized regions of H. americanus eyestalk tissues, we detected peaks attributed to Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe in extracted tissue samples, but not in any eyestalk tissues analyzed directly by MALDI-FTMS. Because methanol is not used as a tissue washing solvent or as a matrix solvent in our protocol for the preparation of direct tissue samples, we felt confident that Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe formation would be prevented during direct tissue analyses. To further explore the possibility that Orc[Ala 11 ] is an endogenous neuropeptide in the H. americanus eyestalk ganglion, we analyzed additional localized SG, LG, XO/MT, MI and ME eyestalk tissue samples dissected from a minimum of three individuals using direct tissue MALDI-FTMS to determine if sampling variability or differences between individuals could be responsible for our inability to detect putative Orc[Ala 11 ]. Furthermore, we collected between three and ten spectra from different regions of each MALDI sample to account for heterogeneity within each sample. In the case of SGs, a source of putative Orc[Ala 11 ] in a previous study, we have collected direct tissue spectra from more than 30 individuals. A representative MALDI-FT mass spectrum from a H. americanus SG gland is shown in Fig. 15A; an expansion of the mass range where Orc[Ala 11 ] would appear (Fig. 15B) reveals no signals characteristic of Orc[Ala 11 ], although other orcokinin family peptides are abundant in the full MALDI-FT mass spectrum. We detected peaks for Orc [1][2][3][4][5][6][7][8][9][10][11] in some, but not all, spectra. In the replicated direct tissue MALDI-FTMS characterizations of localized pieces of eyestalk ganglion tissues from multiple individuals, we failed to detect signals characteristic of Orc[Ala 11 ] in any spectra.
Because Orc[Ala 11 ] has been reported in other H. americanus neuroendocrine organs and tissues, including the supraesophageal ganglion (SoG/brain) [4,30], pericardial organ (PO) [6], and the adult and embryonic stomatogastric ganglion (STG) [4,23], we evaluated the direct tissue MALDI-FT mass spectra of these organs and tissues, as well as H. americanus commissural ganglia (CoG). We again characterized tissues derived from a minimum of three individuals to determine if sampling variability or differences between individuals could be responsible for our inability to detect putative Orc[Ala 11 ]. Furthermore, we collected between three and ten spectra from different regions of each MALDI sample to account for heterogeneity within each sample. For the brain and POs, we also analyzed multiple samples of tissue that were dissected from different locations from the larger sample. CoGs were analyzed in their entirety or split into two pieces prior to analysis, while the entire STG was co-crystallized with matrix. We also characterized the brain from a juvenile lobster. Representative spectra from the tissues analyzed in our laboratory are shown in Fig. 15.
We also examined direct tissue spectra for the STG and CoG, two additional nervous system ganglia. Signals for putative Orc[Ala 11 ] and Orc [1][2][3][4][5][6][7][8][9][10][11] were previously reported in H. americanus STG tissues using direct tissue analyses [4,23], where acidified methanol was used to wash tissue samples and the tissue samples were co-crystallization with DHB in 50% methanol. In our direct tissue investigations of these ganglia, we once again excluded methanol from the sample preparation. A representative spectrum from an STG ( Fig. 15I and J) shows complements of peptides similar to those detected by the Li group [4,23], including an abundant signal from Val 1 -SIF. An examination of the mass range where putative Orc[Ala 11 ] should appear ( Fig. 15I and J) shows three peptides, TNWNKFQGSWamide (m/z 1266.60), pQDLDHVFLRFamide (m/z 1271.65), and STNWSSLRSAWamide (m/z 1293.63), which have been detected in the previous work [4,23]; however, we did not detect signals for Orc[Ala 11 ] in spectra of any STG tissues we examined. We also failed to detect signals for Orc[Ala 11 ] in the MALDI-FTMS spectra for any CoGs (Fig. 15K and L), where we have examined samples from over 20 individuals.

Discussion
For any study aspiring to characterize the endogenous components of a biological system, an underlying assumption is that the sampling and analysis approach will leave the sample in an unaltered state. Our results document a highly specific neuropeptide structural alteration, namely the combined truncation and C-terminal methylation of orcokinin family peptides, that occurs only when biological components of a crustacean tissue sample are present in an acidified methanolic extraction solvent.

The peptide NFDEIDRSGFG-OMe is produced from full-length orcokinin peptides via methanolysis in acidified, methanolic extraction solvents
We used SORI-CID (product ion mass spectra from [M+H] + and y 5 ions) to identify an m/z 1270.57 peptide detected in H. americanus eyestalk tissue extracts as NFDEIDRSGFG-OMe, not isobaric, previously identified NFDEIDRSGFA, based upon the comparisons with standard-derived SORI-CID reference spectra. Furthermore, we used LC/MS to show that the extraction-derived peptide eluted with the same retention time as an Orc[1-11]-OMe, not Orc[Ala 11 ], standard. We used extraction with CD 3 OD to show that methanol from the solvent is the source of the single methyl group found in the m/z 1270.57 peptide detected in H. americanus eyestalk tissue extracts, and applied Q-TOF-CID to show that the added methyl group is found only at the C-terminus of the peptide, Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe. A second truncated, C-terminally methylated peptide, SSEDMDRLGFG-OMe, was also identified based upon mass measurements and labeling experiments with CD 3 OD.

Orc[Ala
To determine if we were able to detect Orc[Ala 11 ] by direct tissue analyses, we analyzed additional eyestalk tissues and other H. americanus neuronal glands and tissues (PO, brain, STG, and CoG) by direct tissue MALDI-FTMS, where methanol is not used in any steps of our tissue preparation protocol. All of our measurements, which often included multiple sub-samples dissected from larger tissues (PO and brain), and which represented measurement from a minimum of three individuals and a maximum of greater than 20 individuals for SG and CoG samples, failed to show any evidence of peaks characteristic of Orc[Ala 11 ] in any spectra.
While it is impossible to prove that a peptide is not present in an organism, our best efforts, using direct tissue analyses and three independent extraction-based approaches, failed to show signals supporting prior work identifying Orc[Ala 11 ] as a peptide endogenous to the lobster. Acidified methanol has been used as the extraction solvent of choice in many previous investigations of invertebrate neuropeptides [1,10,14,21,35,39]. For the extraction of crustacean tissues, research groups have commonly used methanolic solvent systems composed of 90% methanol, 1% water [10,14,21,39] or 9% water [46], and acetic acid. Furthermore, for many MALDIbased analyses of crustacean tissues, acidified methanol has been commonly used as a tissue washing protocol and methanol is a component of the DHB matrix solutions [16,26,29,30].
The truncated orcokinin, Orc[Ala 11 ], was first reported by Huybrechts et al. as a novel peptide de novo sequenced from the Jonah crab, C. borealis [21]. For that study, brain and thoracic ganglion tissues from 50 animals were extracted in methanol:water:acetic acid (90:9:1) and peptides were sequenced using ESI-Q-TOF MS/MS. As was the case for H. americanus, this peptide sequence, with an alanine appearing as the 11th residue, is at odds with the sequences of full-length C. borealis orcokinin peptides, which have been established by many MS studies [21,32]. We suggest that the peptide reported by Huybrechts et al. is, in fact, Orc[1-11]-OMe; this assertion is supported by work carried out in our laboratory, where we have evidence for the detection of Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe in C. borealis brain tissue extracts (data not shown), but not for any directly analyzed C. borealis tissues (CoG, SG, PO, brain) (data not shown). Because alanine (A) is isobaric with methylation at a C-terminal glycine residue (G-OMe), this distinction would not have been revealed from mass measurements. Furthermore, the MS/MS technique used for de novo peptide sequencing in the Huybrechts et al. study would not have provided any obvious flags to distinguish the C-terminal alanine from G-OMe.
The orcokinins NFDEIDRSGFA, SSEDMDRLGFA, and NFDEI-DRSSFA, all with an alanine as the 11th residue and all detected in tissues that had been analyzed following extraction with acidified methanol, have been reported in other publications [15,16,21,[30][31][32]. In summary, our work calls into question the identification of these truncated peptides, which may have Gly-OMe, not Ala, at the C-terminus.

The C-terminal Gly-OMe motif may be misidentified as Ala-OH
A unique issue for the in vitro modification detected in this study is the localized methylation at the C-terminus. Because the Gly-OMe modification is isobaric with Ala-OH, this structural change is not detectable via mass measurements. In this study, where the y 5 fragment ion was isolated and dissociated, the argininecontaining, singly protonated y 5 precursors (protonated RSGFGFA or RSGFG-OMe, respectively) yielded MS/MS spectra sensitive to the methyl-esterified C-terminus. Specifically, we observed a [b 4 +H 2 O] + product ion when the C-terminus had a free carboxyl group (for Orc[Ala 11 ]), and that diagnostic ion was missing when the C-terminus was methyl esterified (for Orc [1][2][3][4][5][6][7][8][9][10][11]-OMe). In contrast, the MS/MS spectra generated on our Q-TOF instrument were insensitive to the structural difference, and this approach could not be used for distinguishing the two peptide sequences. Because MS/MS spectra may not provide the specific, diagnostic information needed to distinguish the peptide sequences, and because standards are not always available, other measures, such as running extraction solvent controls with isotopically labeled solvents, may be needed to distinguish this extraction artifact.

Hypotheses regarding the formation of Orc[1-11]-OMe
Protease-catalyzed reactions have been exploited by chemists to carry out a variety of transformations in nonaqueous solvents [2], including C-terminal peptide esterifications [3,22,33,34]. Most enzymes exploited for this purpose are serine or cysteine proteases, which form reactive acyl-enzyme intermediates that can be attacked by a competing nucleophile, such as methanol.