Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonah crab,Cancer borealis

Mass spectrometric methods were applied to determine the peptidome of the brain and thoracic ganglion of the Jonah crab (Cancer borealis). Fractions obtained by high performance liquid chromatography were characterized using MALDI-TOF MS and ESI-Q-TOF MS/MS. In total, 28 peptides were identified within the molecular mass range 750-3000Da. Comparison of the molecular masses obtained with MALDI-TOF MS with the calculated molecular masses of known crustacean peptides revealed the presence of at least nine allatostatins, three orcokinin precursor derived peptides, namely FDAFTTGFGHS, [Ala(13)]-orcokinin, and [Val(13)]-orcokinin, and two kinins, a tachykinin-related peptide and four FMRFamide-related peptides. Eight other peptides were de novo sequenced by collision induced dissociation on the Q-TOF system and yielded AYNRSFLRFamide, PELDHVFLRFamide or EPLDHVFLRFamide, APQRNFLRFamide, LNPFLRFamide, DVRTPALRLRFamide, and LRNLRFamide, which belong to the FMRFamide related peptide family, as well as NFDEIDRSGFA and NFDEIDRSSFGFV, which display high sequence similarity to peptide sequences within the orcokinin precursor of Orconectes limosus. Our paper is the first (neuro)peptidomic analysis of the crustacean nervous system.     

Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis

The crustacean stomatogastric ganglion (STG) is modulated by both locally released neuroactive compounds and circulating hormones. This study presents mass spectrometric characterization of the complement of peptide hormones present in one of the major neurosecretory structures, the pericardial organs (POs), and the detection of neurohormones released from the POs. Direct peptide profiling of Cancer borealis PO tissues using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) revealed many previously identified peptides, including proctolin, red pigment concentrating hormone (RPCH), crustacean cardioactive peptide (CCAP), several orcokinins, and SDRNFLRFamide. This technique also detected corazonin, a well-known insect hormone, in the POs for the first time. However, most mass spectral peaks did not correspond to previously known peptides. To characterize and identify these novel peptides, we performed MALDI postsource decay (PSD) and electrospray ionization (ESI) MS/MS de novo sequencing of peptides fractionated from PO extracts. We characterized a truncated form of previously identified TNRNFLRFamide, NRNFLRFamide. In addition, we sequenced five other novel peptides sharing a common C-terminus of RYamide from the PO tissue extracts. High K+ depolarization of isolated POs released many peptides present in this tissue, including several of the novel peptides sequenced in the current study.     

A comparison of the neuropeptides from the retrocerebral complex of adult male and female Manduca sexta using MALDI-TOF mass spectrometry

The occurrence of neuropeptides in the retrocerebral complexes of adult male and females of the tobacco hawkmoth, Manduca sexta, was investigated using matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometry (MS), post source decay (PSD) and collision-induced dissociation (CID) MS/MS. From fractions of methanol extracts of corpora cardiaca (CC)/corpora allata (CA), separated by reversed-phase high performance liquid chromatography (RP-HPLC), a total of 11 mass ions were assigned to known peptides from M. sexta. These peptides were adipokinetic hormone (AKH), FLRFamides I, II and III, crustacean cardioactive peptide (CCAP), cardioactive peptide 2b (CAP(2b)), three myoinhibitory peptides, corazonin, and M. sexta allatostatin (Manse-AS). A further six masses were in agreement with Y/FXFGLamide allatostatins identified from other Lepidoptera. The sequence identities of FLRFamide I and AKH were confirmed using post source decay analysis. Fragmentation by collision-induced dissociation MS/MS identified an extended AKH peptide. The apparent differences in the peptides present in male and female retrocerebral complexes are most likely quantitative rather than sex specific.     

Imaging mass spectrometry of neuropeptides in decapod crustacean neuronal tissues

Imaging mass spectrometry (IMS) of neuropeptides in crustacean neuronal tissues was performed on a MALDI-TOF/TOF instrument. Sample preparation protocols were developed for the sensitive detection of these highly complex endogenous signaling molecules. The neuromodulatory complements of the pericardial organ (PO) and brain of the Jonah crab, Cancer borealis, were mapped. Distributions of peptide isoforms belonging to 10 neuropeptide families were investigated using the IMS technique. Often, neuropeptides of high sequence homology were similarly located. However, two RFamide-family peptides and a truncated orcokinin peptide were mapped to locations distinct from other members of their respective families. Over 30 previously sequenced neuropeptides were identified based on mass measurement. For increased confidence of identification, select peptides were fragmented by post-source decay (PSD) and collisional-induced dissociation (CID). Collectively, this organ-level IMS study elucidates the spatial relationships between multiple neuropeptide isoforms of the same family as well as the relative distributions of neuropeptide families.     

Discovery and characterization of the Crustacean hyperglycemic hormone precursor related peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes sapidus using multiple tandem mass spectrometry techniques

The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules-neuropeptides. Via a multifaceted mass spectrometry (MS) approach, 70 neuropeptides were identified including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), pigment dispersing hormone (PDH), proctolin, RFamides, RYamides, and HL/IGSL/IYRamide. Among them, 15 novel orcokinins, 9 novel CPRPs, 1 novel orcomyotropin, 1 novel Ork/Orcomyotropin-related peptide, and 1 novel PDH were de novo sequenced via collision induced dissociation (CID) from the SG of a model organism Callinectes sapidus. Electron transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and higher charge state. Capillary isoelectric focusing (CIEF) was employed for separation of members of the orcokinin family, which is one of the most abundant neuropeptide families observed in the SG. Collectively, our study represents the most complete characterization of neuropeptides in the SG and provides a foundation for future investigation of the physiological function of neuropeptides in the SG of C. sapidus.     

Profiling of neuropeptides released at the stomatogastric ganglion of the crab, Cancer borealis with mass spectrometry

Studies of release under physiological conditions provide more direct data about the identity of neuromodulatory signaling molecules than studies of tissue localization that cannot distinguish between processing precursors and biologically active neuropeptides. We have identified neuropeptides released by electrical stimulation of nerves that contain the axons of the modulatory projection neurons to the stomatogastric ganglion of the crab, Cancer borealis. Preparations were bathed in saline containing a cocktail of peptidase inhibitors to minimize peptide degradation. Both electrical stimulation of projection nerves and depolarization with high K+ saline were used to evoke release. Releasates were desalted and then identified by mass using MALDI-TOF (matrix-assisted laser desorption/ionization-time-of-flight) mass spectrometry. Both previously known and novel peptides were detected. Subsequent to electrical stimulation proctolin, Cancer borealis tachykinin-related peptide (CabTRP), FVNSRYa, carcinustatin-8, allatostatin-3 (AST-3), red pigment concentrating hormone, NRNFLRFa, AST-5, SGFYANRYa, TNRNFLRFa, AST-9, orcomyotropin-related peptide, corazonin, Ala13-orcokinin, and Ser9-Val13-orcokinin were detected. Some of these were also detected after high K+ depolarization. Release was calcium dependent. In summary, we have shown release of the neuropeptides thought to play an important neuromodulatory role in the stomatogastric ganglion, as well as numerous other candidate neuromodulators that remain to be identified.     

Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster, Homarus americanus

Neuropeptides in the stomatogastric ganglion (STG) and the brain of adult and late embryonic Homarus americanus were compared using a multi-faceted mass spectral strategy. Overall, 29 neuropeptides from 10 families were identified in the brain and/or the STG of the lobster. Many of these neuropeptides are reported for the first time in the embryonic lobster. Neuropeptide extraction followed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry enabled confident identification of 24 previously characterized peptides in the adult brain and 13 peptides in the embryonic brain. Two novel peptides (QDLDHVFLRFa and GPPSLRLRFa) were de novo sequenced. In addition, a comparison of adult to embryonic brains revealed the presence of an incompletely processed form of Cancer borealis tachykinin-related peptide 1a (CabTRP 1a, APSGFLGMRG) only in the embryonic brain. A comparison of adult to embryonic STGs revealed that QDLDHVFLRFa was present in the embryonic STG but absent in the adult STG, and CabTRP 1a exhibited the opposite trend. Relative quantification of neuropeptides in the STG revealed that three orcokinin family peptides (NFDEIDRSGFGF, NFDEIDRSGFGFV, and NFDEIDRSGFGFN), a B-type allatostatin (STNWSSLRSAWa), and an orcomyotropin-related peptide (FDAFTTGFGHS) exhibited higher signal intensities in the adult relative to the embryonic STG. RFamide (Arg-Phe-amide) family peptide (DTSTPALRLRFa), [Val¹]SIFamide (VYRKPPFNGSIFa), and orcokinin-related peptide (VYGPRDIANLY) were more intense in the embryonic STG spectra than in the adult STG spectra. Collectively, this study expands our current knowledge of the H. americanus neuropeptidome and highlights some intriguing expression differences that occur during development.     

Identification of neuropeptides from the decapod crustacean sinus glands using nanoscale liquid chromatography tandem mass spectrometry

Neurosecretory systems are known to synthesize and secrete a diverse class of peptide hormones which regulate many physiological processes. The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules--neuropeptides. As an ongoing effort to define the peptidome of the crustacean SG, we determine the neuropeptide complements of the SG of the Jonah crab, Cancer borealis, and the Maine lobster, Homarus americanus, using nanoflow liquid chromatography electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS. Numerous neuropeptides were identified, including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), beta-pigment dispersing hormone (beta-PDH), proctolin and HL/IGSL/IYRamide. Among them, two novel orcokinins were de novo sequenced from the SG of H. americanus. Three CPRPs including a novel isoform were sequenced in H. americanus. Four new CPRPs were sequenced from the SG of C. borealis. Our results show that structural polymorphisms in CPRPs (and thus the CHH precursors) are common in Dendrobranchiata as well as in Pleocyemata. The evolutionary relationship between the CPRPs is also discussed.     

Adipokinetic hormones (AKHs) of sphingid Lepidoptera, including the identification of a second M. sexta AKH

The adipokinetic hormones (AKHs) from the corpora cardiaca (CC) of representative species from all three subfamilies of the Sphingidae (hawkmoths) were investigated using matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) and liquid chromatography electrospray ion trap mass spectrometry (LC-ESI MS), including a re-examination of the AKH complement of the tobacco hawkmoth, Manduca sexta. In addition to larvae and adults of M. sexta (subfamily: Sphinginae), adults from the following subfamilies were examined: Macroglossinae (large elephant hawkmoth, Deilephila elpenor), Smerinthinae (poplar hawkmoth, Laothoe populi and eyed hawkmoth, Smerinthus ocellata), and Sphinginae (death's head hawkmoth, Acherontia atropos). All moths are shown to have the nonapeptide Manse-AKH (pELTFTSSGWamide) in their CC, together with a second AKH, which, on the basis of mass ions ([M+Na](+), [M+K](+)) and partial sequence analysis is identical in all species examined. The structure of this AKH was elucidated from peptides leached out of the CC of adult M. sexta and shown, by ESI-collision-induced dissociation (CID) tandem mass spectrometry (MS/MS), to be a novel decapeptide AKH with a sequence of pELTFSSGWGQamide. The new peptide has been code named Manse-AKH-II. Sequence confirmation was obtained from identical MS studies with synthetic Manse-AKH-II and with the native peptide. Manse-AKH-II has significant lipid-mobilizing activity when injected at low dose (5pmol) into newly emerged adult M. sexta. The potential implications of a second AKH, in M. sexta in particular, are discussed in relation to putative receptor(s).     

Characterization of antimalarial SPf66 peptide using MALDI-TOF MS,CD and SEC

SPf66 is the first chemically synthesized peptide to elicit a partial protective immune response against malaria. Size-exclusion chromatography (SEC) with multi-angle laser light-scattering (MALLS) detection and hydrogen/deuterium (H/D) exchange monitored by (matrix-assisted laser desorption/ionization) MALDI-TOF (time-of-flight) mass spectrometry (MS) were used to assess the conformation and stability in aqueous solution after storage at different temperatures. Moreover, the feasible conformational changes of this peptide were also measured by circular dichroism (CD)-spectroscopy. The absolute molecular weight of SPf66 monomer and dimer species were 4765 and 8960Da using SEC with MALLS detection, and 4643 and 9490Da by MALDI-TOF MS, the discrepancy being between both methods lower than 5.7%, a value quite close to those found in other proteins. The results from H/D exchange monitored by MALDI-TOF MS and CD-spectroscopy show that the SPf66 monomer lacks ordered structure, whereas the SPf66 dimer species presents segments of secondary structure as a determined by CD measurements.     

MALDI-TOF MS of phosphatidylethanolamines: different adducts cause different post source decay (PSD) fragment ion spectra

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly applied to lipids. However, positional acyl chain analysis of lipids by MALDI was so far scarcely described. In this paper, the fragmentation behavior of phosphatidylethanolamine (PE) is investigated by using post-source decay (PSD) MS. In dependence on the investigated adduct, significant differences could be obtained. It will be shown that in particular the negative ion spectra enable the determination of the individual acyl chains as well as their positions (sn-1 or sn-2). Therefore, MALDI-TOF PSD spectra are a real alternative to more sophisticated MS/MS methods.     

Identification of neuropeptides from brains of larval Manduca sexta and Lacanobia oleracea using MALDI-TOF mass spectrometry and post-source decay

The occurrence of neuropeptides in the brain of larvae of the tobacco hawkmoth, Manduca sexta, and tomato moth, Lacanobia oleracea, was investigated using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and post-source decay (PSD). Methanolic extracts of 100 brains separated by reversed-phase high performance liquid chromatography yielded numerous ion peaks, some of which were common to both species. In M. sexta six [M+H](+) ions were in agreement with peptides previously structurally characterised from M. sexta (FLRF-amides I, II and III, M. sexta allatostatin, CAP(2b) and myoinhibitory peptide VI), whereas a further five corresponded to other known lepidopteran peptides (cydiastatins 3 and 4, helicostatins 1 and 6 and helicokinin II). Of these the identities of FLRF-amide I, cydiastatins 3 and 4 and CAP(2b) were confirmed by PSD analysis. Fourteen [M+H](+) ions corresponding to known lepidopteran peptides (FLRF-amide I, cydiastatins 2, 3 and 4, helicostatins 1, 5, 6, 7 and 9, CCAP, CAP(2b), M. sexta allatostatin and myoinhibitory peptide VI) were measured in L. oleracea brain extracts. From this insect, cydiastatins 3 and 4, helicostatin 5 and FLRF-amide I were identified by PSD. These peptides had not previously been structurally characterised from L. oleracea.     

Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.     

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.     

Application of electrospray ionization MS/MS and matrix-assisted laser desorption/ionization-time of flight mass spectrometry to structural analysis of the glycosyl-phosphatidylinositol-anchored protein.

We applied the improved sensitivity and soft ionization characteristics of electrospray Ionization (ESI)-MS/MS and matrix-assisted laser desorption/ionization(MALDI)-time of flight (TOF) mass spectrometry (MS) to analysis of the GPI-anchored C-terminal peptide derived from 5'-nucleotidase. ESI-MS/MS analysis was applied to the core structure (MW, 2,743). In the collision-induced dissociation (CID) spectrum, single-charged ions such as m/z 162 (glucosamine), 286 (mannose-phosphate-ethanolamine), and 447 ([mannose-phosphate-ethanolamine]-glucosamine) were clearly detected as characteristic fragment ions of the GPI-anchored peptide. On MALDI-TOF-MS analysis, heterogeneous peaks of GPI-anchored peptides were detected as single-charged ions in the positive mode. Product ions were obtained by post-source decay (PSD) of m/z 2,905 using curved field reflectron of TOF-MS. Most of the expected product ions derived from the GPI-anchored peptide, containing the core structure and an additional mannose side chain, were successively obtained. Thus, ESI-MS/MS and MALDI-TOF-PSD-MS proved to be effective and sensitive methods for analyzing the GPI-anchored peptide structure with less than 10 pmol of sample. These characteristic fragments or fragmentation patterns seem to be very useful for identification of GPI-anchored C-terminal peptides derived from any kind of GPI-anchored protein.     

Analysis of peptides in the brain and corpora cardiaca-corpora allata of the honey bee, Apis mellifera using MALDI-TOF mass spectrometry

The neuropeptide profiles and diversity of the brain and retrocerebral organs (corpora cardiaca-corpora allata; CC-CA) of adult workers of the honey bee Apis mellifera carnica (dark European strain) were investigated using a combination of HPLC and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with post-source decay (PSD) and collision-induced dissociation (CID) fragmentation. Using evidence from genomic sources, including BLAST searches of the honey bee genome, comparisons with other species and de novo sequencing by PSD and CID fragmentation, a total of 13 mass ions could be assigned to peptides predicted from the A. mellifera genomic database. Peptides positively identified were A. mellifera tachykinin-related peptides 3 and 4 (APMGFQGMRa; APMGFYGTRa) and leucomyosuppressin (pEDVDHVFLRFa). Peptides tentatively identified were A. mellifera tachykinin-related peptides 2 and 5 (ALMGFQGVRa; ARMGFHGMRa), A. mellifera allatostatins 2, 3 and 4 (GRDYSFGLa; RQYSFGLa; GRQPYSFGLa), A1-SIFamide (AYRKPPFNGSIFa), Q1-leucomyosuppressin (QDVDHVFLRFa) and A. mellifera pyrokinins PK 1, PK 2 and Q1-PK 2 (TSQDITSGMWFGPRLa; pEITQFTPRLa; QITQFTPRLa). Allatostatins, tachykinin-related peptides and A1-SIFamide were not detected in CC-CA extract, which appears to contain predominantly leucomyosuppressin, Q1-leucomyosuppressin, PK 1, PK 2, Q1-PK 2 and some unidentified masses. No ion signal was detected that would correspond to the hypertrehalosaemic peptide (=Manse-AKH), which has been isolated from the Italian race of the honey bee (A. mellifera ligustica), but not from A. mellifera carnica.     

Mechanistic insights into the multistage gas-phase fragmentation behavior of phosphoserine- and phosphothreonine-containing peptides

The increasing use of multistage tandem mass spectrometry (MS/MS and MS (3)) methods for comprehensive phosphoproteome analysis studies, as well as the emerging application of in silico spectral intensity prediction algorithms in enhanced database search analysis strategies, necessitate the development of an improved understanding of the mechanisms and other factors that affect the gas-phase fragmentation reactions of phosphorylated peptide ions. To address this need, we have examined the multistage collision-induced dissociation (CID) behavior of a set of singly and doubly charged phosphoserine- and phosphothreonine-containing peptide ions, as well as their regioselectively or uniformly deuterated derivatives, in a quadrupole ion trap mass spectrometer. Consistent with previous reports, the neutral loss of phosphoric acid (H 3PO 4) was observed as a dominant reaction pathway upon MS/MS. The magnitude of this loss was found to be highly dependent on the proton mobility of the precursor ion for both phosphoserine- and phosphothreonine-containing peptides. In contrast to that currently accepted in the literature, however, the results obtained in this study unequivocally demonstrate that the loss of H 3PO 4 does not predominantly occur via a "charge-remote" beta-elimination reaction. The observation of product ions corresponding to the loss of formaldehyde (CH 2O, 30 Da, or CD 2O, 32 Da) or acetaldehyde (CH 3CHO, 44 Da) upon MS (3) dissociation of the [M+ nH-H 3PO 4] ( n+ ) product ions from phosphoserine- and phosphothreonine-containing peptide ions, respectively, provide experimental evidence for a "charge-directed" mechanism involving an S N2 neighboring group participation reaction, resulting in the formation of a cyclic product ion. Potentially, these "diagnostic" MS (3) product ions may provide additional information to facilitate the characterization of phosphopeptides containing multiple potential phosphorylation sites.     

Sequence determination by MALDI-TOF mass spectrometry of an insecticidal lentil peptide of the PA1b type

Mature seeds of lentil (Lens culinaris Medik.) were previously reported to contain an insecticidal cysteine-rich peptide, likely of the albumin-1 subunit b type. The purpose of this work was to determine the amino acid sequence of this insecticidal lentil peptide in an Eston lentil extract by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), after reduction of the disulfide bridges, alkylation of the cysteine residues and hydrolysis by pronase, trypsin, chymotrypsin and endoproteinase Asp-N. Sequences of key fragments were supported by monoisotopic mass measurements and by sequence ions from collision-induced dissociation (CID) experiments with a MALDI-TOF/TOF analyzer (MS/MS analysis). The new 37 amino acid sequence revealed strong similarities to a histidine-containing pea PA1b peptide and to soybean leginsulins but with a unique segment of RSSA in the middle. The lentil PA1b peptide sequence agreed completely with that derived from a L. culinaris genomic DNA sequence.     

Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.     

Effectiveness of CID, HCD, and ETD with FT MS/MS for degradomic-peptidomic analysis: comparison of peptide identification methods

We report on the effectiveness of CID, HCD, and ETD for LC-FT MS/MS analysis of peptides using a tandem linear ion trap-Orbitrap mass spectrometer. A range of software tools and analysis parameters were employed to explore the use of CID, HCD, and ETD to identify peptides (isolated from human blood plasma) without the use of specific "enzyme rules". In the evaluation of an FDR-controlled SEQUEST scoring method, the use of accurate masses for fragments increased the number of identified peptides (by ~50%) compared to the use of conventional low accuracy fragment mass information, and CID provided the largest contribution to the identified peptide data sets compared to HCD and ETD. The FDR-controlled Mascot scoring method provided significantly fewer peptide identifications than SEQUEST (by 1.3-2.3 fold) and CID, HCD, and ETD provided similar contributions to identified peptides. Evaluation of de novo sequencing and the UStags method for more intense fragment ions revealed that HCD afforded more contiguous residues (e.g., ≥ 7 amino acids) than either CID or ETD. Both the FDR-controlled SEQUEST and Mascot scoring methods provided peptide data sets that were affected by the decoy database used and mass tolerances applied (e.g., identical peptides between data sets could be limited to ~70%), while the UStags method provided the most consistent peptide data sets (>90% overlap). The m/z ranges in which CID, HCD, and ETD contributed the largest number of peptide identifications were substantially overlapping. This work suggests that the three peptide ion fragmentation methods are complementary and that maximizing the number of peptide identifications benefits significantly from a careful match with the informatics tools and methods applied. These results also suggest that the decoy strategy may inaccurately estimate identification FDRs.