Mass Spectrometry Study and Infrared Spectroscopy of the Complex Between Camphor and the Two Enantiomers of Protonated Alanine: The Role of Higher‐Energy Conformers in the Enantioselectivity of the Dissociation Rate Constants

The properties of the protonated complexes built from S camphor and R or S alanine were studied in a Paul ion trap at room temperature by collision‐induced dissociation (CID) and infrared multiple‐photon dissociation spectroscopy (IRMPD), as well as molecular dynamics and ab initio calculations. While the two diastereomer complexes display very similar vibrational spectra in the fingerprint region, in line with similar structures, and almost identical calculated binding energies, their collision‐induced dissociation rates are different. Comparison of the IRMPD results to computed spectra shows that the SS and SR complexes both contain protonated alanine strongly hydrogen‐bonded to the keto group of camphor. The floppiness of this structure around the NH⁺…O = C hydrogen bond results in a complex potential energy surface showing multiple minima. Calculating the dissociation rate constant within the frame of the transition state theory shows that the fragmentation rate larger for the heterochiral SR complex than the homochiral SS complex can be explained in terms of two almost isoenergetic low‐energy conformers in the latter that are not present for the former. Chirality 25:436‐443, 2013. © 2013 Wiley Periodicals, Inc.     

De novo peptide sequencing using CID and HCD spectra pairs

In tandem mass spectrometry (MS/MS), there are several different fragmentation techniques possible, including, collision‐induced dissociation (CID) higher energy collisional dissociation (HCD), electron‐capture dissociation (ECD), and electron transfer dissociation (ETD). When using pairs of spectra for de novo peptide sequencing, the most popular methods are designed for CID (or HCD) and ECD (or ETD) spectra because of the complementarity between them. Less attention has been paid to the use of CID and HCD spectra pairs. In this study, a new de novo peptide sequencing method is proposed for these spectra pairs. This method includes a CID and HCD spectra merging criterion and a parent mass correction step, along with improvements to our previously proposed algorithm for sequencing merged spectra. Three pairs of spectral datasets were used to investigate and compare the performance of the proposed method with other existing methods designed for single spectrum (HCD or CID) sequencing. Experimental results showed that full‐length peptide sequencing accuracy was increased significantly by using spectra pairs in the proposed method, with the highest accuracy reaching 81.31%.     

PTM MarkerFinder,a software tool to detect and validate spectra from peptides carrying post‐translational modifications

Mass spectrometry (MS) analysis of peptides carrying post‐translational modifications is challenging due to the instability of some modifications during MS analysis. However, glycopeptides as well as acetylated, methylated and other modified peptides release specific fragment ions during CID (collision‐induced dissociation) and HCD (higher energy collisional dissociation) fragmentation. These fragment ions can be used to validate the presence of the PTM on the peptide. Here, we present PTM MarkerFinder, a software tool that takes advantage of such marker ions. PTM MarkerFinder screens the MS/MS spectra in the output of a database search (i.e., Mascot) for marker ions specific for selected PTMs. Moreover, it reports and annotates the HCD and the corresponding electron transfer dissociation (ETD) spectrum (when present), and summarizes information on the type, number, and ratios of marker ions found in the data set. In the present work, a sample containing enriched N‐acetylhexosamine (HexNAc) glycopeptides from yeast has been analyzed by liquid chromatography‐mass spectrometry on an LTQ Orbitrap Velos using both HCD and ETD fragmentation techniques. The identification result (Mascot .dat file) was submitted as input to PTM MarkerFinder and screened for HexNAc oxonium ions. The software output has been used for high‐throughput validation of the identification results.     

Analysis of sesterterpenoids from Aspergillus terreus using ESI‐QTOF and ESI‐IT

Introduction – Biosynthesis of terretonin was studied due to the interesting skeleton of this series of sesterterpenoids. Very recently, López‐Gresa reported two new sesterterpenoids (terretonins E and F) which are inhibitors of the mammalian mitochondrial respiratory chain. Mass spectrometry (MS), especially tandem mass spectrometry, has been one of the most important physicochemical methods for the identification of trace natural products due to it rapidity, sensitivity and low levels of sample consumption. The potential application prospect and unique skeleton prompted us to study structural characterisation using MS. Objective – To obtain sufficient information for rapid structural elucidation of this class of compounds using MS. Methodology – The elemental composition of the product ions was confirmed by low‐energy ESI‐CID‐QTOF‐MS/MS analyses. The fragmentation pathways were postulated on the basis of ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ spectra. Common features and major differences between ESI‐QTOF‐MS/MS and IT‐MSⁿ spectra were compared. For ESI‐QTOF‐MS/MS/MS experiments, capillary exit voltage was raised to induce in‐source dissociation. Ammonium acetate or acetic acid were added into solutions to improve the intensity of [M + H]⁺. The collision energy was optimised to achieve sufficient fragmentation. Some fragmentation pathways were unambiguously proposed by the variety of abundance of fragment ions at different collision energies even without MSⁿ spectra. Results – Fragmentation pathways of five representative sesterterpenoids were elucidated using ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ in both positive‐ and negative‐ion mode. The key group of characterising fragmentation profiles was ring B, and these fragmentation patterns are helpful to identify different types of sestertepenoids. Conclusion – Complementary information obtained from fragmentation experiments of [M + H]⁺ (or [M + NH₄]⁺) and [M ? H]^? precursor ions is especially valuable for rapid identification of this kind of sesterterpenoid.     

RNA‐sequencing profiles hippocampal gene expression in a validated model of cancer‐induced depression

To investigate the pathophysiology of cancer‐induced depression (CID), we have recently developed a validated CID mouse model. Given that the efficacy of antidepressants in cancer patients is controversial, it remains unclear whether CID is a biologically distinct form of depression. We used RNA‐sequencing (RNA‐seq) to investigate differentially expressed genes (DEGs) in hippocampi of animals from our CID model relative a positive control model of depressive‐like behavior induced with chronic corticosterone (CORT). To validate RNA‐seq results, we performed quantitative real‐time RT‐PCR (qRT‐PCR) on a subset of DEGs. Enrichment analysis using DAVID was performed on DEGs to identify enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and biological process gene ontologies (GO:BP). qRT‐PCR results significantly predicted RNA‐seq results. RNA‐seq revealed that most DEGs identified in the CORT model overlapped with the CID model. Enrichment analyses identified KEGG pathways and GO:BP terms associated with ion homeostasis and neuronal communication for both the CORT and CID model. In addition, CID DEGs were enriched in pathways and terms relating to neuronal development, intracellular signaling, learning and memory. This study is the first to investigate CID at the mRNA level. We have shown that most hippocampal mRNA changes that are associated with a depressive‐like state are also associated with cancer. Several other changes occur at the mRNA level in cancer, suggesting that the CID model may represent a biologically distinct form of a depressive‐like state.     

C-terminal sequencing by mass spectrometry: application to gelatine-derived proline-rich peptides

Protonated peptides derived from proline‐rich proteins (PRP) are often difficult to sequence by standard collision‐induced dissociation (CID) mass spectrometry (MS) due to preferential amide bond cleavage N‐terminal to proline. In connection with bovine spongiform encephalopathy regulations, proteolytic products derived from the PRP collagen have been suggested as markers for contamination of animal feedstuffs with processed animal protein (Fernandez Ocaña, M. et al., Analyst 2004, 129, 111–115). Herein, we report the identification of these marker peptides using the strategy of C‐terminal sequencing by CID MS from their sodium and lithium adducts. Upon fragmentation a new cationized peptide was produced that is one C‐terminal amino acid shorter in length. This dissociation pathway allowed for the facile identification of the C‐terminal residue by matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry. Each newly formed cationized peptide was further fragmented by up to seven stages of electrospray ionization ion trap MS. Proline‐rich C‐terminal sequence tags were established which permitted successful database identification of collagen alpha type I proteins.     

Studies on the Selective Trifluoroacetolytic Scission of Native Peptaibols and Model Peptides Using HPLC and ESI‐CID‐MS

Representative members of a group of linear, N‐acylated polypeptide antibiotics (peptaibols) containing α‐aminoisobutyric acid (Aib) and, in part, isovaline (Iva), as well as proteinogenic amino acids and a C‐terminal‐bonded 2‐amino alcohol, were treated with anhydrous trifluoroacetic acid (TFA) at 37° for 0.5–26 h. The resulting fragments were separated by HPLC and characterized by electrospray ionization collision‐induced dissociation mass spectrometry (ESI‐CID‐MS). The following 16–20‐residue peptaibols were investigated: natural, microheterogeneous mixtures of antiamoebins and alamethicin F50, uniform paracelsin A, and synthetic trichotoxin A50/E. In the natural peptides, bonds formed between Aib (Iva) and Pro (Hyp) were rapidly and selectively cleaved within 0.5 h. Furthermore, TFA esters of the C‐terminal amino alcohols were formed. Depending on time, release of C‐terminal tri‐ and tetrapeptides as well as amino acids from the major fragments was observed. Synthetic homooligopeptides, namely Z‐ and Ac‐(Aib)₁₀‐O^tBu and Z‐(Aib)₇‐O^tBu, were analyzed for comparison. On treatment with TFA, a regular series of Z‐(Aib)_10–5‐OH from Z‐(Aib)₁₀‐O^tBu were detected within 0.5 h, and, after 3 h, release of a regular series of Z‐(Aib)_7–3‐OH from Z‐(Aib)₇‐O^tBu were observed. Moreover, concomitant release of the series of H‐(Aib)_10–3‐OH from the decapeptide occurred. From these data, a repetitive cleavage mechanism via intermediate formation of C‐terminal oxazolones on trifluoroacetolysis is proposed. Furthermore, their formation and stability in native peptaibols are correlated with subtle structural differences in protein amino acids linked to Aib. From the conspicuous concordance of the formation and abundance of regular series of trifluoroacetolytic fragments and of positive ions of the b‐series in CID‐MS, the generation of intermediate oxazolonium ions in both gas and liquid phase is concluded.     

Identification and characterization of citrulline‐modified brain proteins by combining HCD and CID fragmentation

Citrullination is a protein PTM of arginine residues catalyzed by peptidylarginine deiminase. Protein citrullination has been detected in the CNS and associated with a number of neurological diseases. However, identifying citrullinated proteins from complex mixtures and pinpointing citrullinated residues have been limited. Using RP LC and high‐resolution MS, this study determined in vitro citrullination sites of glial fibrillary acid protein (GFAP), neurogranin (NRGN/RC3), and myelin basic protein (MBP) and in vivo sites in brain protein extract. Human GFAP has five endogenous citrullination sites, R30, R36, R270, R406, and R416, and MBP has 14 in vivo citrullination sites. Human NRGN/RC3 was found citrullinated at residue R68. The sequence of citrullinated peptides and citrullination sites were confirmed from peptides identified in trypsin, Lys‐C, and Glu‐C digests. The relative ratio of citrullination was estimated by simultaneous identification of citrullinated and unmodified peptides from Alzheimer's and control brain samples. The site occupancy of citrullination at the residue R68 of NRGN ranged from 1.6 to 9.5%. Compared to CID, higher‐energy collisional dissociation (HCD) mainly produced protein backbone fragmentation for citrullinated peptides. CID‐triggered HCD fragmentation is an optimal approach for the identification of citrullinated peptides in complex protein digests.     

Ion trap collisional activation of c and z* ions formed via gas-phase ion/ion electron-transfer dissociation

A series of c- and z*-type product ions formed via gas-phase electron-transfer ion/ion reactions between protonated polypeptides with azobenzene radical anions are subjected to ion trap collision activation in a linear ion trap. Fragment ions including a-, b-, y-type and ammonia-loss ions are typically observed in collision induced dissociation (CID) of c ions, showing almost identical CID patterns as those of the C-terminal amidated peptides consisting of the same sequences. Collisional activation of z* species mainly gives rise to side-chain losses and peptide backbone cleavages resulting in a-, b-, c-, x-, y-, and z-type ions. Most of the fragmentation pathways of z* species upon ion trap CID can be accounted for by radical driven processes. The side-chain losses from z* species are different from the small losses observed from the charge-reduced peptide molecular species in electron-transfer dissociation (ETD), which indicates rearrangement of the radical species. Characteristic side-chain losses are observed for several amino acid residues, which are useful to predict their presence in peptide/protein ions. Furthermore, the unique side-chain losses from leucine and isoleucine residues allow facile distinction of these two isomeric residues.     

Integrating titania enrichment,iTRAQ labeling,and Orbitrap CID‐HCD for global identification and quantitative analysis of phosphopeptides

Recent advances in MS instrumentation and progresses in phosphopeptide enrichment, in conjunction with more powerful data analysis tools, have facilitated unbiased characterization of thousands of site‐specific phosphorylation events. Combined with stable isotope labeling by amino acids in cell culture metabolic labeling, these techniques have made it possible to quantitatively evaluate phosphorylation changes in various physiological states in stable cell lines. However, quantitative phosphoproteomics in primary cells and tissues remains a major technical challenge due to the lack of adequate techniques for accurate quantification. Here, we describe an integrated strategy allowing for large scale quantitative profiling of phosphopeptides in complex biological mixtures. In this technique, the mixture of proteolytic peptides was subjected to phosphopeptide enrichment using a titania affinity column, and the purified phosphopeptides were subsequently labeled with iTRAQ reagents. After further fractionation by strong‐cation exchange, the peptides were analyzed by LC‐MS/MS on an Orbitrap mass spectrometer, which collects CID and high‐energy collisional dissociation (HCD) spectra sequentially for peptide identification and quantitation. We demonstrate that direct phosphopeptide enrichment of protein digests by titania affinity chromatography substantially improves the efficiency and reproducibility of phosphopeptide proteomic analysis and is compatible with downstream iTRAQ labeling. Conditions were optimized for HCD normalized collision energy to balance the overall peptide identification and quantitation using the relative abundances of iTRAQ reporter ions. Using this approach, we were able to identify 3557 distinct phosphopeptides from HeLa cell lysates, of which 2709 were also quantified from HCD scans.     

O‐glycoside sequence of pentacyclic triterpene saponins from Phytolacca bogotensis using HPLC‐ESI/multi‐stage tandem mass spectrometry

Introduction – The lack of pharmacopoeial methodologies for the quality control of plants used for therapeutic purposes is a huge problem that impacts directly upon public health. In the case of saponins, their great structural complexity, weak glycoside bonds and high polarity hinder their identification by conventional techniques. Objective – To apply high‐performance liquid chromatography–electrospray tandem mass spectrometry (HPLC‐ESI/MSⁿ) to identify the O‐glycoside sequence of saponins from the roots of Phytolacca bogotensis. Methodology – Saponins were isolated by preparative HPLC and characterised by NMR spectroscopic experiments. Collision‐induced dissociation (CID) of isolated saponins was performed producing typical degradation reactions that can be associated with several glycosidic bonds as empirical criteria. A method using solid‐phase extraction (SPE) and HPLC/ESI‐MSⁿ for the characterisation of saponins and identification of novel molecules is described. Results – Three saponins reported for the first time in P. bogotensis were isolated and characterised by NMR spectroscopy. Characteristic cross ring cleavage reactions have been used as empirical criteria for the characterisation of the glycosidic bonds most frequently reported for Phytolacca saponins. One new saponin was proposed on the basis of empirical criteria, and other five saponins were identified for the first time for P. bogotensis using HPLC‐ESI/MSⁿ. Conclusion – Electrospray ionisation in combination with tandem mass spectrometry has been established as a powerful tool for the profiling of saponins from roots of P. bogotensis. CID proved to be a useful tool for the characterisation and identification of known and novel saponins from the plant family Phytolaccaceae and can be used for quality control purposes of crude plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

Combining low- and high-energy tandem mass spectra for optimized peptide quantification with isobaric tags

Isobaric tagging, via TMT or iTRAQ, is widely used in quantitative proteomics. To date, tandem mass spectrometric analysis of isobarically-labeled peptides with hybrid ion trap–orbitrap (LTQ-OT) instruments has been mainly carried out with higher-energy C-trap dissociation (HCD) or pulsed q dissociation (PQD). HCD provides good fragmentation of the reporter-ions, but peptide sequence-ion recovery is generally poor compared to collision-induced dissociation (CID). Herein, we describe an approach where CID and HCD spectra are combined. The approach ensures efficiently both identification and relative quantification of proteins. Tandem mass tags (TMTs) were used to label digests of human plasma and LC-MS/MS was performed with an LTQ-OT instrument. Different HCD collision energies were tested. The benefits to use CID and HCD with respect to HCD alone were demonstrated in terms of number of identifications, subsequent number of quantifiable proteins, and quantification accuracy. A program was developed to merge the peptide sequence-ion m/z range from CID spectra and the reporter-ion m/z range from HCD spectra, and alternatively to separate both spectral data into different files. As parallel CID in the LTQ almost doesn't affect the analysis duty cycle, the procedure should become a standard for quantitative analyses of proteins with isobaric tagging using LTQ-OT instruments.     

Molecular mapping of stomatal‐conductance‐related traits in durum wheat (Triticum turgidum ssp. durum)

The most promising traits identified in wheat to raise yield potential via an increase in biomass accumulation are stomatal conductance and stomatal‐conductance‐related traits, such as carbon isotope discrimination (CID) and photosynthetic rate. The evaluation of the extent of genetic variation and the mapping of chromosomal regions controlling these traits are essential for the development of effective breeding strategies in durum wheat. A population of 161 F2‐derived, F8–F9 recombinant inbred lines obtained from a cross between durum wheat (Triticum turgidum ssp. durum) cultivars Ofanto and Cappelli was phenotyped for heading date, plant height, leaf porosity, CID and chlorophyll concentration (estimated through the SPAD index) for 2007/2008 and 2008/2009 seasons, at Ottava, Sardinia (Italy) under irrigated conditions. The genotype mean heritability for leaf porosity, CID and chlorophyll concentration was moderate in size. Six quantitative trait loci were detected for leaf porosity, four for chlorophyll concentration, but only one for CID, because of the small variation expressed in the population for this trait under these experimental conditions. The quantitative trait loci for leaf porosity located on chromosome 3B appear to be more stable with respect to the others, and different microsatellite markers are positioned within the interval of the quantitative trait loci, or in their vicinity, that represent useful tools in programmes for selection assisted by molecular markers.     

Detection of a phosphorylated glycine-serine linker in an IgG-based fusion protein

Molecular mass determination by electrospray ionization mass spectrometry of a recombinant IgG-based fusion protein (mAb1-F) produced in human embryonic kidney (HEK) cells demonstrated the presence of a dominant +79 Da product variant. Using LC-MS tryptic peptide mapping analysis and collision-induced dissociation (CID) and electron-transfer/higher-energy collision dissociation fragmentations, the modification was localized to the C-terminal serine residue of a glycine-serine linker [(G₄S)₂] of a fused heavy chain containing in total 2 (G₄S)₂-linkers. The modification was identified as a phosphorylation (+79.97 Da) by the presence of a 98 Da neutral loss reaction with CID, by spiking a synthetic phosphoserine peptide, and by dephosphorylation with alkaline phosphatase. A thermolysin digest combined with higher-energy collision dissociation (HCD) positioned the phosphoserine to one specific glycine-serine linker of the fused heavy chain, and the relative level of phosphorylated linker was determined to be 11.3% and 0.4% by LC-MS when the fusion protein was transiently expressed in HEK or in stably transformed Chinese hamster ovary cells, respectively. This observation demonstrates that fusions with glycine-serine linker sequences should be carefully evaluated during drug development to prevent the introduction of a phosphorylation site in therapeutic fusion proteins.     

Evidence for specific subunit distribution and interactions in the quaternary structure of α‐crystallin

The quaternary structure of α‐crystallin is dynamic, a property which has thwarted crystallographic efforts towards structural characterization. In this study, we have used collision‐induced dissociation mass spectrometry to examine the architecture of the polydisperse assemblies of α‐crystallin. For total α‐crystallin isolated directly from fetal calf lens using size‐based chromatography, the αB‐crystallin subunit was found to be preferentially dissociated from the oligomers, despite being significantly less abundant overall than the αA‐crystallin subunits. Furthermore, upon mixing molar equivalents of purified αA‐ and αB‐crystallin, the levels of their dissociation were found to decrease and increase, respectively, with time. Interestingly though, dissociation of subunits from the αA‐ and αB‐crystallin homo‐oligomers was comparable, indicating that strength of the αA:αA, and αB:αB subunit interactions are similar. Taken together, these data suggest that the differences in the number of subunit contacts in the mixed assemblies give rise to the disproportionate dissociation of αB‐crystallin subunits. Limited proteolysis mass spectrometry was also used to examine changes in protease accessibility during subunit exchange. The C‐terminus of αA‐crystallin was more susceptible to proteolytic attack in homo‐oligomers than that of αB‐crystallin. As subunit exchange proceeded, proteolysis of the αA‐crystallin C‐terminus increased, indicating that in the hetero‐oligomeric form this tertiary motif is more exposed to solvent. These data were used to propose a refined arrangement for the interactions of the α‐crystallin domains and C‐terminal extensions of subunits within the α‐crystallin assembly. In particular, we propose that the palindromic IPI motif of αB‐crystallin gives rise to two orientations of the C‐terminus. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos

Over the past decade peptide sequencing by collision induced dissociation (CID) has become the method of choice in mass spectrometry-based proteomics. The development of alternative fragmentation techniques such as electron transfer dissociation (ETD) has extended the possibilities within tandem mass spectrometry. Recent advances in instrumentation allow peptide fragment ions to be detected with high speed and sensitivity (e.g., in a 2D or 3D ion trap) or at high resolution and high mass accuracy (e.g., an Orbitrap or a ToF). Here, we describe a comprehensive experimental comparison of using ETD, ion-trap CID, and beam type CID (HCD) in combination with either linear ion trap or Orbitrap readout for the large-scale analysis of tryptic peptides. We investigate which combination of fragmentation technique and mass analyzer provides the best performance for the analysis of distinct peptide populations such as N-acetylated, phosphorylated, and tryptic peptides with up to two missed cleavages. We found that HCD provides more peptide identifications than CID and ETD for doubly charged peptides. In terms of Mascot score, ETD FT outperforms the other techniques for peptides with charge states higher than 2. Our data shows that there is a trade-off between spectral quality and speed when using the Orbitrap for fragment ion detection. We conclude that a decision-tree regulated combination of higher-energy collisional dissociation (HCD) and ETD can improve the average Mascot score.     

Isolation,purification and de novo sequencing of TBD‐1, the first beta‐defensin from leukocytes of reptiles

A novel peptide with antimicrobial activity was isolated from leukocytes of the European pond turtle Emys orbicularis and purified to homogeneity by preparative gel electrophoresis followed by reversed phase chromatography. It was highly active in vitro against Escherichia coli, Listeria monocytogenes, methicillin‐resistant Staphylococcus aureus, and Candida albicans. The isolated peptide was sequenced de novo by tandem mass spectrometry using both collision‐induced and electron‐transfer dissociation in combination with different chemical derivatization techniques. The 40‐residue peptide, called TBD‐1 (turtle β‐defensin 1), represents the first defensin isolated from reptilian leukocytes. It contains three disulfide bonds and shows high structural similarities to β‐defensins isolated from birds and mammals.     

Comparison of vacuum matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI (AP-MALDI) tandem mass spectrometry of 2-dimensional separated and trypsin-digested glomerular proteins for database search derived identification

Mass spectrometric based sequencing of enzymatic generated peptides is widely used to obtain specific sequence tags allowing the unambiguous identification of proteins. In the present study, two types of desorption/ionization techniques combined with different modes of ion dissociation, namely vacuum matrix-assisted laser desorption/ionization (vMALDI) high energy collision induced dissociation (CID) and post-source decay (PSD) as well as atmospheric pressure (AP)-MALDI low energy CID, were applied for the fragmentation of singly protonated peptide ions, which were derived from two-dimensional separated, silver-stained and trypsin-digested hydrophilic as well as hydrophobic glomerular proteins. Thereby, defined properties of the individual fragmentation pattern generated by the specified modes could be observed. Furthermore, the compatibility of the varying PSD and CID (MS/MS) data with database search derived identification using two public accessible search algorithms has been evaluated. The peptide sequence tag information obtained by PSD and high energy CID enabled in the majority of cases an unambiguous identification. In contrast, part of the data obtained by low energy CID were not assignable using similar search parameters and therefore no clear results were obtainable. The knowledge of the properties of available MALDI-based fragmentation techniques presents an important factor for data interpretation using public accessible search algorithms and moreover for the identification of two-dimensional gel separated proteins.     

Complementary structural information from a tryptic N-linked glycopeptide via electron transfer ion/ion reactions and collision-induced dissociation

Glycosylation is an important post-translational modification. Analysis of glycopeptides is difficult using collision-induced dissociation, as it typically yields only information about the glycan structure, without any peptide sequence information. We demonstrate here how a 3D-quadrupole ion trap, using the complementary techniques of collision induced dissociation (CID) and electron-transfer dissociation (ETD), can be used to elucidate the glycan structure and peptide sequence of the N-glycosylated peptide from a fractionated tryptic digest of the lectin from the coral tree, Erythina cristagalli. CID experiments on the multiply protonated glycopeptide ions yield, almost exclusively, cleavage at glycosidic bonds, with little peptide backbone fragmentation. ETD reactions of the triply charged glycopeptide cations with either sulfur dioxide or nitrobenzene anions yield cleavage of the peptide backbone with no loss of the glycan structure. These results show that a 3D-quadrupole ion trap can be used to provide glycopeptide amino acid sequence information as well as information about the glycan structure.