De novo sequencing of novel neuropeptides directly from Ascaris suum tissue using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight

Direct analysis of tissue by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows for the rapid profiling of biological molecules with minimal loss of sample or degradation and reduced likelihood of chemical modification. However, there are still considerable challenges to overcome due to the complexity of tissue and the low quantity of endogenous peptide in a single cell. These problems are exacerbated in the nematode Ascaris suum because of the small size of individual neurons and the paucity of peptide per cell. In an effort to address these difficulties, the recently developed matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) technology was used in combination with an on-target derivatization in order to sequence novel neuropeptides directly from Ascaris nervous tissue. Direct MALDI-TOF/TOF analysis of Ascaris tissue provided the complete amino acid sequences for a previously characterized neuropeptide as well as for three novel peptides with homologues found in other nematodes. These results demonstrate a method for the rapid characterization of sub-femtomolar amounts of peptide directly from tissue using MALDI-TOF/TOF.     

Peptidomic analysis of the larval Drosophila melanogaster central nervous system by two-dimensional capillary liquid chromatography quadrupole time-of-flight mass spectrometry

Peptides are the largest class of signalling molecules found in animals. Nevertheless, in most proteomic studies peptides are overlooked since they literally fall through the mazes of the net. In analogy with proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomic approach aims at the simultaneous visualization and identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their post-translational modifications. In this paper we describe the analysis of the larval fruit fly central nervous system using two-dimensional capillary liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS/MS. Using the central nervous systems of only 50 larval Drosophila as starting material, we identified 38 peptides in a single analysis, 20 of which were not detected in a previous study that reported on the one-dimensional capillary LC/MS/MS analysis of the same tissue. Among the 38 sequenced peptides, some originate from precursors, such as the tachykinin and the IFamide precursor that were entirely missed in the first study. This clearly demonstrates that the two-dimensional capillary LC approach enhances the coverage of the peptidomic analysis.     

On-tissue protein identification and imaging by MALDI-Ion mobility mass spectrometry

MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for the detection and localization of drugs, proteins, and lipids on-tissue. Nevertheless, this approach can only perform identification of low mass molecules as lipids, pharmaceuticals, and peptides. In this article, a combination of approaches for the detection and imaging of proteins and their identification directly on-tissue is described after tryptic digestion. Enzymatic digestion protocols for different kinds of tissues—formalin fixed paraffin embedded (FFPE) and frozen tissues—are combined with MALDI-ion mobility mass spectrometry (IM-MS). This combination enables localization and identification of proteins via their related digested peptides. In a number of cases, ion mobility separates isobaric ions that cannot be identified by conventional MALDI time-of-flight (TOF) mass spectrometry. The amount of detected peaks per measurement increases (versus conventional MALDI-TOF), which enables mass and time selected ion images and the identification of separated ions. These experiments demonstrate the feasibility of direct proteins identification by ion-mobility-TOF IMS from tissue. The tissue digestion combined with MALDI-IM-TOF-IMS approach allows a proteomics “bottom-up” strategy with different kinds of tissue samples, especially FFPE tissues conserved for a long time in hospital sample banks. The combination of IM with IMS marks the development of IMS approaches as real proteomic tools, which brings new perspectives to biological studies.     

Three dimensional mapping of neuropeptides and lipids in crustacean brain by mass spectral imaging

Imaging mass spectrometry is emerging as a powerful tool that has been applied extensively for the localization of proteins, peptides, pharmaceutical compounds, metabolites, and lipids in biological tissues. In this article, a three-dimensional mass spectral imaging (3D MSI) technique was developed to examine distribution patterns of multiple neuropeptide families and lipids in the brain of the crab Cancer borealis. Different matrix/solvent combinations were compared for preferential extraction and detection of neuropeptides and lipids. Combined with morphological information, the distribution of numerous neuropeptides throughout the 3D structure of brain was determined using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Different localization patterns were observed for different neuropeptide families, and isoforms displaying unique distribution patterns that were distinct from the common family distribution trends were also detected. In addition, multiple lipids were identified and mapped from brain tissue slices. To confirm their identities, MS/MS fragmentation was performed. Different lipid species displayed distinct localization patterns, suggesting their potential different functional roles in the nervous system.     

Toward a high-throughput approach to quantitative proteomic analysis: Expression-dependent protein identification by mass spectrometry

The isotope-coded affinity tag (ICAT) [1] technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry. The initial implementation of this technology was based on microcapillary chromatography coupled on-line with electrospray ionization tandem mass spectrometry. This implementation lacked the ability to select proteins for identification based on their relative abundance and therefore to focus on differentially expressed proteins. In order to improve the sample throughput of this technology, we have developed a two-step approach that is focused on those proteins for which the abundance changes between samples: First, a new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance and second, these peptides are identified by tandem mass spectrometry using an electrospray quadrupole time-of flight mass spectrometer and sequence database searching. Results from the application of this approach to the analysis of differentially expressed proteins secreted from nontumorigenic human prostate epithelial cells and metastatic cancerous human prostate epithelial cells are shown.     

A strategy for distinguishing modified peptides based on post-digestion 18O labeling and mass spectrometry

The simultaneous identification of multiple different protein modifications, with or without known mass changes, is a challenging application of mass spectrometry. In this contribution, a strategy for distinguishing modified peptides within a large background of unmodified peptides was demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of cytochrome c (Cyt-c) modified with 4-hydroxy-2-nonenal (HNE), based on post-digestion 18O labeling. Labeling of control Cyt-c peptides obtained from in-solution or in-gel digestion with 18O, prior to mixing in the ratio of 1:1 with peptides derived from a modified sample, identified more HNE modifications than a method based on a known mass increment search (Isom AL, Barnes S, Wilson L, Kirk M, Coward L, Darley-Usmar V. J. Am. Soc. Mass Spectrom. 2004; 15: 1136), demonstrating the potential of this strategy to enhance the detection of modified peptides by mass spectrometry. A virtue of the strategy is that it obviates the need for isotopic labeling of the modifier, making the method applicable to the detection of modifications occurring in vivo. Additionally, this technique identified protease auto-cleavage peptides by their altered mass isotopomer distribution due to incomplete 18O exchange, and modified peptides containing 'protein carbonyls' by partial 18O exchange, allowing these peptides to be differentiated during data analysis.     

Determination of species-specific components in the venom of Parabuthus scorpions from southern Africa using matrix-assisted laser desorption time-of-flight mass spectrometry

The aim of the present study was to analyze mass spectra of scorpions belonging to the genus Parabuthus (Pocock 1890) by means of matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOFMS) and to construct a species-specific venom code for species identification. The venom compositions of sixteen Parabuthus species, occurring in southern Africa, were characterized using representative peaks in the molecular mass range of 6400-8400 Da. This mass range is characteristic for the typical long-chain neurotoxins influencing sodium channels. Only a few of these peptides have been sequenced up to now. The impetus for development of these species-specific profiles was the observation of unique, highly reproducible mass spectral peaks within a specific species. An identification label for all the different species could be found using a minimum number of peaks. MALDI-TOFMS is therefore proposed as a complementary method to morphological and behavioural characteristics for species and ultimately subspecies discrimination.     

Characterization of peptides from Aplysia using microbore liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry guided purification

Liquid chromatography (LC) has been used extensively for the separation and isolation of peptides due to its high selectivity and peak capacity. An approach combining microbore LC with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) detection is described to identify peptides in cells and guide the purification of peptides from the marine mollusc Aplysia californica. Direct MALDI-MS of neurons and processes provides molecular mass information for unknown peptides with almost no sample preparation, and LC-MALDI-MS allows the isolation and purification of these peptides from pooled samples, thus enabling new putative neuropeptides to be isolated from complex cellular samples. Both direct MALDI-MS and LC-MALDI-MS are compared in terms of detecting peptides from neuronal samples. Using both approaches, two peaks from Aplysia californica connectives having molecular masses of 5013 and 5021 have been isolated, partially sequenced and identified as novel collagen-like peptides.     

Electrospray ionization quadrupole time-of-flight and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometric analyses to solve micro-heterogeneity in post-translationally modified peptides from Phoneutria nigriventer (Aranea, Ctenidae) venom

Previous studies of the fractionated venom of the Brazilian armed spider Phoneutria nigriventer, obtained by gel filtration, have demonstrated the presence of a fraction PhM, a pool of small peptides (up to 2000 Da) that provoke contractions in smooth muscle of guinea pig ileum. Initial attempts to sequence these peptides were largely unsuccessful because of the low purification yield and the fact that the majority seemed to be blocked at their N-termini. In the present work, analysis of this venom fraction by mass spectrometry has revealed the existence of a highly complex mixture of peptides with molecular weights corresponding to those observed for the muscle-active peptides previously described (800-1800 Da). These peptides appear to be a family of isoforms with some particular features. The amino acid sequences of 15 isoforms have been determined by tandem mass spectrometry (MS/MS) using both electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q/ToFMS) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToFMS). These molecules contain post-translational modifications such as proteolysis and C-terminal amidation, which combine to generate additional isoforms. All the isoforms sequenced in this study possess an N-terminal pyroglutamic acid residue. A search for sequence similarities with other peptides in databanks revealed that these peptides are structurally related to the tachykinins, a family of neuro-hormone peptides. The data obtained in this study will be essential for the subsequent steps of this research, the synthesis of these peptides and pharmacological characterization of their biological activity.     

Identification of bioactive peptides in hypoallergenic infant milk formulas by capillary electrophoresis-mass spectrometry

In this study, we use capillary electrophoresis-mass spectrometry (CE-MS) for the identification of bioactive peptides in hypoallergenic infant milk formulas (IF), which are complex bovine milk protein hydrolysates. A sample clean-up pretreatment with a citrate buffer containing dithiothreitol and urea followed by solid-phase extraction (SPE) with different reversed-phase commercial cartridges was investigated to achieve optimum detection sensitivity in CE-MS. SPE with C18, StrataX and Oasis HLB cartridges allowed detection of the largest number of low molecular mass components, but combination of C18 and StrataX results was enough to achieve an excellent coverage of the studied IF. The monoisotopic molecular mass values of the low molecular mass components obtained by capillary electrophoresis ion-trap mass spectrometry (CE-IT-MS) allowed the tentative identification of nine bioactive sequences. Only the identification of five of them could be confirmed when accurate mass measurements were performed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS), namely LKP, IPY, ALPM, PGPIHN and VAGTWY, which were reported to present angiotensin-converting enzyme (ACE) inhibitory and antimicrobial activity (only VAGTWY).     

Nanospray analysis of the venom of the tarantula Theraphosa leblondi: a powerful method for direct venom mass fingerprinting and toxin sequencing

Mass spectrometric methods, including matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), on-line liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS), and nanospray ionisation/hybrid quadrupole time-of-flight mass spectrometry (nanoESI-QqTOFMS), were applied to characterize by mass fingerprinting the venom of the French Guyanese tarantula Theraphosa leblondi. Of these techniques direct nanoESI-QqTOFMS, which allowed the detection of 65 protonated molecules with high mass accuracy, appeared to give the best results. Three major peptides, TlTx1, TlTx2 and TlTx3, were sequenced using a combination of nanoESI-MS/MS and enzyme digestion/MS and MS/MS experiments. Each sequence was confirmed by automated Edman sequencing. In patch-clamp experiments these peptides were found to have a specific inhibitory effect on the voltage-dependent potassium channel, Kv4.2.     

Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.     

Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry for Rapid Determination of the Metabolites of Baicalin Produced by Human Intestinal Bacteria

Baicalin, mainly isolated from Scutllaria baicalensis, has been reported to possess a wide range of biological activities. However, the information about the metabolic route and metabolites of baicalin was limited to the role of the human intestinal bacterial mixture. In this paper, four strains of bacteria including Bacteroides sp. 33 and 56, and Veillonella sp. 23 and 71 were isolated from human intestinal bacterial mixture and studied for their abilities to convert baicalin to different metabolites. A highly sensitive and specific ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) method combined with mass defect filtering (MDF) provides high throughput capabilities for drug metabolism study. The chromatographic separation was performed on a 1.7 µm particle size C 18 column using gradient elution system. The components in the extract were identified and confirmed according to the mass spectrometric fragmentation mechanisms, MS/MS fragment ions and relevant literature by means of electrospray ionization mass spectrometry in negative ion mode. With this method, a total of 4 metabolites were identified based on MS and MS/MS data. The results indicated that hydrogenation, methylation, and deglycosylation were the major metabolic pathways of baicalin in vitro. The present study provides important information about the metabolism of baicalin which will be helpful for fully understanding the impact of the intestinal bacteria on this active component. Furthermore, this work demonstrated the potential of the ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry approach for a rapid, simple, reliable, and automated identification of metabolites of natural products.     

N-terminal H3/D3-acetylation for improved high-throughput peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer

A novel method for peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) is presented. A stable isotope label introduced in the peptide N-terminus by derivatization, using a 1:1 mixture of acetic anhydride and deuterated acetic anhydride, allows for easy and unambiguous identification of ions belonging either to the N- or the C-terminal ion series in the product ion spectrum, making sequence assignment significantly simplified. The good performance of this technique was shown by successful sequencing of the contents of several peptide maps. A similar approach was recently applied to nanoelectrospray ionization (nanoESI) and nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS). The MALDI-TOF/TOF technique allows for fast, direct sequencing of modified peptides in proteomics samples, and is complementary to the nanoESI and nanoLC/MS/MS approaches.     

Characterization of phosphopeptides from protein digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray quadrupole time-of-flight mass spectrometry

A two-step mass spectrometric method for characterization of phosphopeptides from peptide mixtures is presented. In the first step, phosphopeptide candidates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) based on their higher relative intensities in negative ion MALDI spectra than in positive ion MALDI spectra. The detection limit for this step was found to be 18 femtomoles or lower in the case of unfractionated in-solution digests of a model phosphoprotein, beta-casein. In the second step, nanoelectrospray tandem mass (nES-MS/MS) spectra of doubly or triply charged precursor ions of these candidate phosphopeptides were obtained using a quadrupole time-of-flight (Q-TOF) mass spectrometer. This step provided information about the phosphorylated residues, and ruled out nonphosphorylated candidates, for these peptides. After [(32)P] labeling and reverse-phase high-performance liquid chromatography (RP-HPLC) to simplify the mixtures and to monitor the efficiency of phosphopeptide identification, we used this method to identify multiple autophosphorylation sites on the PKR-like endoplasmic reticulum kinase (PERK), a recently discovered mammalian stress-response protein.     

High-throughput one-bead-one-compound approach to peptide-encoded combinatorial libraries: MALDI-MS analysis of single TentaGel beads

The identification of pharmacologically promising compounds (lead compounds) from combinatorial libraries is frequently limited by the throughput of the analytical technique employed. Fourier transform mass spectrometry (FTMS) offers high sensitivity, mass accuracy (m/Deltam > 500 000), and sequencing capabilities. A rapid and efficient method for high-throughput analysis of single beads from peptide-encoded combinatorial libraries with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is presented. Encoding peptides on single beads are identified and structurally characterized by MALDI time-of-flight (TOF) and ultrahigh-resolution MALDI Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. A strategy of on-probe sample preparation is developed to minimize handling of the beads.     

Rapid characterization of Bacillus spores targeting species-unique peptides produced with an atmospheric pressure matrix-assisted laser desorption/ionization source

New and improved strategies are eagerly sought for the rapid identification of microorganisms, particularly in mixtures. Mass spectrometry remains a powerful tool for this purpose. Small acid-soluble proteins (SASPs), which are relatively abundant in Bacillus spores, represent potential biomarkers for species characterization. Despite sharing extensive sequence homology, these proteins differ sufficiently in sequence for discrimination between species. This work focuses on the differences in sequence between SASPs from various Bacillus species. Compilation of SASP sequences from protein database searches, followed by in silico trypsin digestion and analysis of the resulting fragments, identified several species-specific peptides that could be targeted for analysis using mass spectrometry. This strategy was tested and found to be successful in the characterization of Bacillus spores both from individual species and in mixtures. Analysis was performed using an ion trap mass spectrometer with an atmospheric pressure MALDI source. This instrumentation offers the advantage of increased speed of analysis and accurate precursor ion selection for tandem mass spectrometric analysis compared with vacuum matrix-assisted laser desorption/ionization and time-of-flight instruments. The identification and targeting of species-specific peptides using this type of instrumentation offers a rapid, efficient strategy for the identification of Bacillus spores and can potentially be applied to different microorganisms.     

Top-down identification of endogenous peptides up to 9 kDa in cerebrospinal fluid and brain tissue by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry

Recent work on protein and peptide biomarker patterns revealed the difficulties in identifying their molecular components, which is indispensable for validation of the biological context. Cerebrospinal fluid and brain tissue are used as sources to discover new biomarkers, e.g. for neurodegenerative diseases. Many of these biomarker candidates are peptides with a molecular mass of <10 kDa. Their identification is favourably achieved with a 'top-down' approach, because this requires less purification and an enzymatic cleavage will often not yield enough specific fragments for successful database searches. Here, we describe an approach using quadrupole time-of-flight mass spectrometry (TOFMS) as a highly efficient mass spectrometric purification and identification tool after off-line decomplexation of biological samples by liquid chromatography. After initial peptidomic screening with matrix-assisted laser desorption/ionization (MALDI) TOFMS, the elution behaviour in chromatography and the exact molecular mass were used to locate the same signals in nanoelectrospray measurements. Most of the peaks detected in MALDI-TOFMS could be retrieved in nanoelectrospray quadrupole TOFMS. Suitable collision energies for informative fragment spectra were investigated for different parent ions, charge states and molecular masses. After collision-induced dissociation, the resulting fragmentation data of multiply charged ions can become much more complicated than those derived from tryptic peptide digests. However, the mass accuracy and resolution of quadrupole TOF instruments results in high-quality data suitable for determining peptide sequences. The protein precursor, proteolytic processing and post-translational modifications were identified by automated database searches. This is demonstrated by the exemplary identifications of thymosin beta-4 (5.0 kDa) and NPY (4.3 kDa) from rat hypothalamic tissue and ubiquitin (8.6 kDa) from human cerebrospinal fluid. The high data quality should also allow for de novo identification. This methodology is generally applicable for peptides up to a molecular mass of about 10 kDa from body fluids, tissues or other biological sources.     

Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO red staining

The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting. Proteins which are not chemically modified during the SYPRO Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS. The validity of SYPRO Red staining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C. albicans proteins from a total of 31 selected protein spots. The peptide mass signal intensity, the number of matched peptides and the percentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modified silver protein gel staining. This work demonstrates that fluorescent SYPRO Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver staining. As far as we know, this is the first report in which C. albicans proteins separated using 2-D gels have been identified by peptide mass fingerprinting. The improved technique described here should be very useful for carrying out proteomic studies.