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1.
Dong-Gi Mun Rohit Budhraja Firdous A. Bhat Roman M. Zenka Kenneth L. Johnson Abhay Moghekar Akhilesh Pandey 《Proteomics》2023,23(10):2200507
A quadrupole time-of-flight mass spectrometer coupled with a trapped ion mobility spectrometry (timsTOF) operated in parallel accumulation-serial fragmentation (PASEF) mode has recently emerged as a platform capable of providing four-dimensional (4D) features comprising of elution time, collision cross section (CCS), mass-to-charge ratio, and intensity of peptides. The PASEF mode provides ∼100% ion sampling efficiency both in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes without sacrificing sensitivity. In addition, targeted measurements using PASEF integrated parallel reaction monitoring (PRM) mode have also been described. However, only limited number of studies have used timsTOF for analysis of clinical samples. Although Orbitrap mass spectrometers have been used for biomarker discovery from cerebrospinal fluid (CSF) in a variety of neurological diseases, these Orbitrap-derived datasets cannot readily be applied for driving experiments on timsTOF mass spectrometers. We generated a catalog of peptides and proteins in human CSF in DDA mode on a timsTOF mass spectrometer and used these data to build a spectral library. This strategy allowed us to use elution times and ion mobility values from the spectral library to design PRM experiments for quantifying previously discovered biomarkers from CSF samples in Alzheimer's disease. When the same samples were analyzed using a DIA approach combined with a spectral library search, a higher number of proteins were identified than in a library-free approach. Overall, we have established a spectral library of CSF as a resource and demonstrated its utility for PRM and DIA studies, which should facilitate studies of neurological disorders. 相似文献
2.
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. 相似文献
3.
Li Cao Nikola Tolić Yi Qu Da Meng Rui Zhao Qibin Zhang Ronald J. Moore Erika M. Zink Mary S. Lipton Ljiljana Paša-Tolić Si Wu 《Analytical biochemistry》2014
Simultaneous elucidation of the glycan structure and the glycosylation site are needed to reveal the biological function of protein glycosylation. In this study, we employed a recent type of fragmentation termed higher energy collisional dissociation (HCD) to examine fragmentation patterns of intact glycopeptides generated from a mixture of standard glycosylated proteins. The normalized collisional energy (NCE) value for HCD was varied from 30 to 60% to evaluate the optimal conditions for the fragmentation of peptide backbones and glycoconjugates. Our results indicated that HCD with lower NCE values preferentially fragmented the sugar chains attached to the peptides to generate a ladder of neutral loss of monosaccharides, thereby enabling the putative glycan structure characterization. In addition, detection of the oxonium ions enabled unambiguous differentiation of glycopeptides from non-glycopeptides. In contrast, HCD with higher NCE values preferentially fragmented the peptide backbone and, thus, provided information needed for confident peptide identification. We evaluated the HCD approach with alternating NCE parameters for confident characterization of intact N- and O-linked glycopeptides in a single liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. In addition, we applied a novel data analysis pipeline, so-called GlycoFinder, to form a basis for automated data analysis. Overall, 38 unique intact glycopeptides corresponding to eight glycosylation sites (six N-linked and two O-linked sites) were confidently identified from a standard protein mixture. This approach provided concurrent characterization of both the peptide and the glycan, thereby enabling comprehensive structural characterization of glycoproteins in a single LC–MS/MS analysis. 相似文献
4.
The archaea Methanococcus maripaludis strain Mm900 produces flagella that are glycosylated with an N-linked tetrasaccharide. Mass spectrometric analysis of flagellar tryptic peptides identified a number of tryptic glycopeptides carrying a glycan of mass 1036.4 Da, and fragmentation of the glycan oxonium ion indicated that the glycan was a tetrasaccharide. The glycan was purified, following extensive pronase digestion of flagellar filaments, by size-exclusion and anion-exchange chromatography. NMR spectroscopy revealed that the glycan had the following structure:Sug-4-β-ManNAc3NAmA6Thr-4-β-GlcNAc3NAcA-3-β-GalNAc-Asnwhere Sug is a novel monosaccharide unit, (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. This oligosaccharide has significant similarity to the oligosaccharide that was found previously in Methanococcus voltae. 相似文献
5.
Wuhrer M Catalina MI Deelder AM Hokke CH 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,849(1-2):115-128
Next to the identification of proteins and the determination of their expression levels, the analysis of post-translational modifications (PTM) is becoming an increasingly important aspect in proteomics. Here, we review mass spectrometric (MS) techniques for the study of protein glycosylation at the glycopeptide level. Enrichment and separation techniques for glycoproteins and glycopeptides from complex (glyco-)protein mixtures and digests are summarized. Various tandem MS (MS/MS) techniques for the analysis of glycopeptides are described and compared with respect to the information they provide on peptide sequence, glycan attachment site and glycan structure. Approaches using electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) of glycopeptides are presented and the following fragmentation techniques in glycopeptide analysis are compared: collision-induced fragmentation on different types of instruments, metastable fragmentation after MALDI ionization, infrared multi-photon dissociation, electron-capture dissociation and electron-transfer dissociation. This review discusses the potential and limitations of tandem mass spectrometry of glycopeptides as a tool in structural glycoproteomics. 相似文献
6.
Barbara Steigenberger Henk W.P. van den Toorn Emiel Bijl Jean-Franois Greisch Oliver Rther Markus Lubeck Roland J. Pieters Albert J.R. Heck Richard A. Scheltema 《Molecular & cellular proteomics : MCP》2020,19(10):1677-1687
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- •Cross-linked peptides are physically separated from mono-linked peptides in the gas-phase by TIMS ion mobility.
- •Development of a novel data acquisition routine that a-priori distinguishes cross-linked from mono-linked peptides called caps-PASEF.
- •First application of PhoX-driven cross-linking mass spectrometry on the timsTOF Pro.
- •Application of cross-linking mass spectrometry to medium to high complexity samples.
7.
Mark L. Stolowitz 《Proteomics》2012,12(23-24):3438-3450
Over the course of the last decade, a number of investigators have come to appreciate that the surface of a MALDI target, after suitable modification, can be used for selective enrichment of peptides and proteins. More recently, surface‐modified nanoparticles (NPs) that readily co‐crystallize in MALDI matrix, are not ionized by laser desorption/ionization, and do not interfere with MS have attracted interest as alternatives to surface‐modified targets for selective enrichment of peptides and proteins. Surface‐modified targets and NPs facilitate parallel processing of samples, and when used in conjunction with MALDI mass spectrometers with kHz lasers enable development of high‐throughput proteomics platforms. Targets and NPs for reversed phase and ion exchange retention, selective enrichment of glycopeptides, selective enrichment of phosphopeptides, and immunoaffinity MS are described in conjunction with details regarding their preparation and utility. Commercial availability of the reagents and substrates required to prepare surface‐modified targets and NPs is also discussed. 相似文献
8.
Hägglund P Bunkenborg J Elortza F Jensen ON Roepstorff P 《Journal of proteome research》2004,3(3):556-566
Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins. 相似文献
9.
Wang H Wong CH Chin A Taguchi A Taylor A Hanash S Sekiya S Takahashi H Murase M Kajihara S Iwamoto S Tanaka K 《Nature protocols》2011,6(3):253-269
We present a protocol for the identification of glycosylated proteins in plasma followed by elucidation of their individual glycan compositions. The study of glycoproteins by mass spectrometry is usually based on cleavage of glycans followed by separate analysis of glycans and deglycosylated proteins, which limits the ability to derive glycan compositions for individual glycoproteins. The methodology described here consists of 2D HPLC fractionation of intact proteins and liquid chromatography-multistage tandem mass spectrometry (LC-MS/MS(n)) analysis of digested protein fractions. Protein samples are separated by 1D anion-exchange chromatography (AEX) with an eight-step salt elution. Protein fractions from each of the eight AEX elution steps are transferred onto the 2D reversed-phase column to further separate proteins. A digital ion trap mass spectrometer with a wide mass range is then used for LC-MS/MS(n) analysis of intact glycopeptides from the 2D HPLC fractions. Both peptide and oligosaccharide compositions are revealed by analysis of the ion fragmentation patterns of glycopeptides with an intact glycopeptide analysis pipeline. 相似文献
10.
Jifeng Zhang Daniel I. C. Wang 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,712(1-2)
A chromatographic method was developed for quantitative analysis of site-specific microheterogeneity of the two N-linked glycosylation sites in recombinant human interferon-γ produced from Chinese hamster ovary (CHO) cell culture. After the interferon-γ was harvested by affinity chromatography, the tryptic digestion was carried out. The two glycopeptide pools, isolated from reversed-phase chromatography of tryptic digestion of interferon-γ, were subjected to further separation by hydrophilic interaction chromatography. Each peak in the chromatograms was identified by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI–TOF–MS). The overall elution order of the glycopeptides was the following: neutral glycopeptides, monosialylated glycopeptides, bisialylated glycopeptides, trisialylated glycopeptide and tetrasialylated glycopeptides. Based on the integrated peak area for each compound in the chromatograms, the percentage for each glycan was utilized to quantify the glycosylation pattern of the interferon-γ. Finally, sialylation and antennarity structure percentages at the two glycosylation sites were chosen as the quality indicators in process monitoring of interferon-γ production from a serum-free suspension-batch CHO culture. 相似文献
11.
A new hydrophilic interaction chromatography (HILIC) column packed with amide 1.7 μm sorbent was applied to the characterization of glycoprotein digests. Due to the impact of the hydrophilic carbohydrate moiety, glycopeptides were more strongly retained on the column and separated from the remaining nonglycosylated peptides present in the digest. The glycoforms of the same parent peptide were also chromatographically resolved and analyzed using ultraviolet and mass spectrometry detectors. The HILIC method was applied to glyco-profiling of a therapeutic monoclonal antibody and proteins with several N-linked and O-linked glycosylation sites. For characterization of complex proteins with multiple glycosylation sites we utilized 2D LC, where RP separation dimension was used for isolation of glycopeptides and HILIC for resolution of peptide glycoforms. The analysis of site-specific glycan microheterogeneity was illustrated for the CD44 fusion protein. 相似文献
12.
Sergey Y. Vakhrushev James Langridge Iain Campuzano Chris Hughes Jasna Peter-Katalinić 《Clinical proteomics》2008,4(1-2):47-57
Introduction
A novel approach of ion mobility tandem mass spectrometry (IMS-MS/MS) is applied to analysis of human glycourinome to obtain carbohydrate pattern data of congenital disorders of glycosylation patient. Overlapping of the complex carbohydrate mass range landscape has been highly reduced upon IMS-MS procedure, allowing more efficient identification by mapping and sequencing of glycan precursor ions, following their separation by mobility, according to difference in drift time through the traveling wave IMS cell. Intact and truncated N- and O-glycan structures modified by sialylation and fucosylation were identified according to their drift time separated molecular ions and submitted to fragmentation in a narrow mass window.IMS CID MS/MS Analysis
The fragmentation spectra generated from the IMS separated precursor ions contain series of fragment ions maintaining the same mobility as their parent ions, and the assignment accuracy can be significantly enhanced.Conclusion
According to the specific fragment ion patterns, carbohydrate epitopes described to be involved in pathological processes were assigned. A high potential of this glycomics-based strategy for clinical applications can be presented. 相似文献13.
Large scale mass spectrometry analysis of N-linked glycopeptides is complicated by the inherent complexity of the glycan structures. Here, we evaluate a mass spectrometry approach for the targeted analysis of N-linked glycopeptides in complex mixtures that does not require prior knowledge of the glycan structures or pre-enrichment of the glycopeptides. Despite the complexity of N-glycans, the core of the glycan remains constant, comprising two N-acetylglucosamine and three mannose units. Collision-induced dissociation (CID) mass spectrometry of N-glycopeptides results in the formation of the N-acetylglucosamine (GlcNAc) oxonium ion and a [mannose+GlcNAc] fragment (in addition to other fragments resulting from cleavage within the glycan). In ion-trap CID, those ions are not detected due to the low m/z cutoff; however, they are detected following the beam-type CID known as higher energy collision dissociation (HCD) on the orbitrap mass spectrometer. The presence of these product ions following HCD can be used as triggers for subsequent electron transfer dissociation (ETD) mass spectrometry analysis of the precursor ion. The ETD mass spectrum provides peptide sequence information, which is unobtainable from HCD. A Lys-C digest of ribonuclease B and trypsin digest of immunoglobulin G were separated by ZIC-HILIC liquid chromatography and analyzed by HCD product ion-triggered ETD. The data were analyzed both manually and by search against protein databases by commonly used algorithms. The results show that the product ion-triggered approach shows promise for the field of glycoproteomics and highlight the requirement for more sophisticated data mining tools. 相似文献
14.
The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides. 相似文献
15.
Jonas Nilsson 《Glycoconjugate journal》2016,33(3):261-272
The use of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MSn) for the glycoproteomic characterization of glycopeptides is a growing field of research. The N- and O-glycosylated peptides (N- and O-glycopeptides) analyzed typically originate from protease-digested glycoproteins where many of them are expected to be biomedically important. Examples of LC-MS2 and MS3 fragmentation strategies used to pursue glycan structure, peptide identity and attachment-site identification analyses of glycopeptides are described in this review. MS2 spectra, using the CID and HCD fragmentation techniques of a complex biantennary N-glycopeptide and a core 1 O-glycopeptide, representing two examples of commonly studied glycopeptide types, are presented. A few practical tips for accomplishing glycopeptide analysis using reversed-phase LC-MSn shotgun proteomics settings, together with references to the latest glycoproteomic studies, are presented. 相似文献
16.
Multiple reaction monitoring (MRM) is a highly sensitive and increasingly popular method of targeted mass spectrometry (MS) that can be used to selectively detect and quantify peptides and their corresponding proteins of interest within biological samples. The sensitivity of MRM-MS is highly dependent upon the tuning of transition-specific parameters, especially the collision energy (CE) applied during peptide fragmentation. Currently, empirical equations for CE work best for y-type ions and are much less effective for other types of transitions, such as b-type ions and small y-type transitions across particular amide bonds, which could also be useful for MRM-MS if optimized for maximum signal transmission. In this work, we have performed a CE optimization of all transitions for 80 doubly charged peptides, the results of which were used to define separate CE equations for b-ions and y-ions, as well as for small y-type ions derived from the fragmentation of amide bonds bounded on the amino-terminal side by aspartic or glutamic acid residues (D/E-X transitions). This analysis yielded four major observations: (1) b-ions tend to require lower collision energies than y-ions for optimal fragmentation, while D/E-X transitions tend to require more; (2) CE equations predict the optimal CEs more closely when product ion m/z dependence is included, in addition to the current standard of precursor ion m/z dependence; (3) separate CE equations for y-ions, b-ions, and D/E-X transitions are more effective than the previous one-size-fits-all equations, but best results are achieved by optimizing transitions individually; and (4) while b-ions gain substantial signal from CE optimization-often increases of several-fold-they still tend to rank lower than y-ions from the same peptide. These results confirm the notion that y-ions are usually the first-choice transitions for MRM experiments but also demonstrate, for the first time, that b-ions can be viable targets as well, if the proper collision energies are used. 相似文献
17.
Abstract Hope rests on the envelope proteins of human immunodeficiency virus (HIV) as protective vaccines and thus their antibody binding sites are of prime interest. 2G12 and other human antibodies bind to a cluster of oligomannose N-glycans. Owing to the extreme number and density of N-glycosylation sites gp160 and its recombinant form gp140 represent challenging tasks for site-specific glycosylation analysis. We have conducted a glycosylation analysis of CN54gp140 by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) using an ion trap as well as a Q-TOF instrument and standard software for glycopeptide identification. First, a deglycosylated sample of the protease digest served to locate the elution positions of peptides covering all of the 27 potential N-glycosylation sites. Then, the assignments of the similarly eluting glycopeptides were verified by collision-induced decay MS/MS experiments with elevated fragmentation energy. The acquisition of site-specific glycan profiles was facilitated by the use of buffered eluent, which rounds up all glycoforms of a peptide into one peak. Calculation of the molecular mass drawn on the weighted averages of the glycans at each site led to the actual mass of gp140 of approximately 120 kDa. 相似文献
18.
Mass spectrometry (MS) has the potential to revolutionize structural glycobiology and help in the understanding of how post-translation events such as glycosylation affect protein activities. Several approaches to determine the structure of glycopeptides have been used successfully including fast atom bombardment, matrix-assisted laser desorption ionization, and electrospray ionization with a wide variety of mass analyzers. However, the identification of glycopeptides in a complex mixture still remains a challenge. The source of this challenge is primarily due to the poor ionization efficiency and rapid degradation of glycopeptides. In this report we describe the use of a chip-based infusion nanoelectrospray ionization technique in combination with a recently developed linear ion trap for identification and characterization of glycosylation in complex mixtures. Two standard synthetic glycans were analyzed using multiple-stage fragmentation analysis in both positive and negative ionization modes. In addition, the high mannose type N-glycosylation in ribonuclease B (RNase B) was used to map the glycosylation site and obtain the glycan structures. We were able to map the glycosylation site and obtain the glycan structures in RNase B in a single analysis. The results reported here demonstrate that the fully automated chip-based nanoelectrospray linear ion trap platform is a valuable system for oligosaccharide analyses due to the unique MS/MS and MS(n) capability of the linear ion trap and the extended analysis time provided by the ionization technique. 相似文献
19.
Halim A Nilsson J Rüetschi U Hesse C Larson G 《Molecular & cellular proteomics : MCP》2012,11(4):M111.013649
Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS(2), Hex(5)HexNAc(4)-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e.g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS(3), of selected fragment-ions from the CID-MS(2) analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan- and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where "intact" glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt. 相似文献
20.
Paolo Nanni Christian Panse Peter Gehrig Susanne Mueller Jonas Grossmann Ralph Schlapbach 《Proteomics》2013,13(15):2251-2255
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. 相似文献