Characterization Of Glycopeptides Research Articles

RIP3 regulates doxorubicin-induced intestinal mucositis via FUT2-mediated α-1,2-fucosylation.

Intestinal mucositis is one of the common side effects of anti-cancer chemotherapy. However, the molecular mechanisms involved in mucositis development remain incompletely understood. In this study, we investigated the function of receptor-interacting protein kinase 3 (RIP3/RIPK3) in regulating doxorubicin-induced intestinal mucositis and its potential mechanisms. Intestinal mucositis animal models were induced in mice for in vivo studies. Rat intestinal cell line IEC-6 was used for in vitro studies. RNA‑seq was used to explore the transcriptomic changes in doxorubicin-induced intestinal mucositis. Intact glycopeptide characterization using mass spectrometry was applied to identify α-1,2-fucosylated proteins associated with mucositis. Doxorubicin treatment increased RIP3 expression in the intestine and caused severe intestinal mucositis in the mice, depletion of RIP3 abolished doxorubicin-induced intestinal mucositis. RIP3-mediated doxorubicin-induced mucositis did not depend on mixed lineage kinase domain-like (MLKL) but on α-1,2-fucosyltransferase 2 (FUT2)-catalyzed α-1,2-fucosylation on inflammation-related proteins. Deficiency of MLKL did not affect intestinal mucositis, whereas inhibition of α-1,2-fucosylation by 2-deoxy-D-galactose (2dGal) profoundly attenuated doxorubicin-induced inflammation and mucositis. RIP3-FUT2 pathway is a central node in doxorubicin-induced intestinal mucositis. Targeting intestinal RIP3 and/or FUT2-mediated α-1,2-fucosylation may provide potential targets for preventing chemotherapy-induced intestinal mucositis.

Direct and Detailed Site-Specific Glycopeptide Characterization by Higher-Energy Electron-Activated Dissociation Tandem Mass Spectrometry.

Glycosylation is widely recognized as the most complex post-translational modification due to the widespread presence of macro- and microheterogeneities, wherein its biological consequence is closely related to both the glycosylation sites and the glycan fine structures. Yet, efficient site-specific detailed glycan characterization remains a significant analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform, higher-energy electron-activated dissociation (heExD) tandem mass spectrometry (MS/MS) revealed extraordinary efficacy for the structural characterization of intact glycopeptides. HeExD produced extensive fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments (with or without the glycan). The intensity of cross-ring cleavage and backbone fragments retaining the intact glycan was highly dependent on the electron energy. Among the four electron energy levels investigated, electronic excitation dissociation (EED) provided the most comprehensive structural information, yielding a complete series of glycosidic fragments for accurate glycan topology determination, a wealth of cross-ring fragments for linkage definition, and the most extensive peptide backbone fragments for accurate peptide sequencing and glycosylation site localization. The glycan fragments observed in the EED spectrum correlated well with the fragmentation patterns observed in EED MS/MS of the released glycans. The advantages of EED over higher-energy collisional dissociation (HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy collisional dissociation (EThcD) were demonstrated for the characterization of a glycopeptide bearing a biantennary disialylated glycan. EED can produce a complete peptide backbone and glycan sequence coverage even for doubly protonated precursors. The exceptional performance of heExD MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization on an Orbitrap-Omnitrap hybrid instrument presents a novel option for in-depth glycosylation analysis.

Chemical Proteomic Approach for In-Depth Glycosylation Profiling of Plasma Carcinoembryonic Antigen in Cancer Patients

Carcinoembryonic antigen (CEA) of human plasma is a biomarker of many cancer diseases, and its N-glycosylation accounts for 60% of molecular mass. It is highly desirable to characterize its glycoforms for providing additional dimension of features to increase its performance in prognosis and diagnosis of cancers. However, to systematically characterize its site-specific glycosylation is challenging because of its low abundance. Here, we developed a highly sensitive strategy for in-depth glycosylation profiling of plasma CEA through chemical proteomics combined with multienzymatic digestion. A trifunctional probe was utilized to generate covalent bond of plasma CEA and its antibody upon UV irradiation. As low as 1 ng/ml CEA in plasma could be captured and digested with trypsin and chymotrypsin for intact glycopeptide characterization. Twenty six of 28 potential N-glycosylation sites were well identified, which were the most comprehensive N-glycosylation site characterization of CEA on intact glycopeptide level as far as we known. Importantly, this strategy was applied to the glycosylation analysis of plasma CEA in cancer patients. Differential site-specific glycoforms of plasma CEA were observed in patients with colorectal cancers (CRCs) and lung cancer. The distributions of site-specific glycoforms were different as the progression of CRC, and most site-specific glycoforms were overexpressed in stage II of CRC. Overall, we established a highly sensitive chemical proteomic method to profile site-specific glycosylation of plasma CEA, which should generally applicable to other well-established cancer glycoprotein biomarkers for improving their cancer diagnosis and monitoring performance.

Assessing the hydrophobicity of glycopeptides using reversed-phase liquid chromatography and tandem mass spectrometry

Retention time is one of the most important parameters that has been widely used to demonstrate the separation results obtained from liquid chromatography (LC) platforms. However, retention time can shift when samples are tested with different instruments and laboratories, which hinders the identification process of analytes when comparing data collected from different LC systems. To address this problem, hydrophobicity index was introduced for retention time normalization of the glycopeptides separated by reversed-phase LC (RPLC). Tandem MS was used for the detection and identification of glycopeptides. In addition, the influence of different types of glycans on the hydrophobicity of peptide backbones was studied by comparing the retention time of glycopeptides with their non-glycosylated counterparts. The hydrophobicity of tryptic digested glycopeptides derived from model glycoproteins, including bovine fetuin, α1-acid glycoprotein, and haptoglobin from human plasma, were evaluated based on the hydrophobicity index of the standard peptides from a peptide retention time calibration mixture. The reduction of hydrophobicity of multiple peptide backbones was observed due to the hydrophilic glycan structures. By comparing the hydrophobicity index of glycopeptides collected from different time and instruments, the day-to-day and lab-to-lab comparisons suggested high reliability and reproducibility of this approach. The RSD% of hydrophobicity index from inter-lab experiments was 1.2%, while the RSD% of retention time was 5.1%. Then, the applications of this method were demonstrated on complex glycopeptide samples extracted from human blood serum. The hydrophobicity index can be applied to address the retention time shift when using different instruments, thereby boosting confidence of the characterization of glycopeptides.

Glycopeptide characterization of Sf9-derived SARS-CoV-2 spike protein recombinant vaccine candidates expedited by the use of glycopeptide libraries.

We report the N-glycosylation pattern of Sf9 insect cell-derived recombinant spike proteins being developed as candidate vaccine antigens for SARS-CoV-2 (COVID-19) (Sanofi). The method has been optimised to produce peptides with single, isolated glycosylation sites using multiple protease digests. The development and use of glycopeptide libraries from previous developmental phases allowed for faster analysis than processing datasets from individual batches from first principles. Purified spike proteins were reduced, alkylated, and digested with proteolytic enzymes. Three different protease digests were utilised to generate peptides with isolated glycosylation sites. The glycopeptides were then analysed using a Waters Q-TOF while using a data-dependent acquisition mass spectrometry experiment. Glycopeptide mapping data processing and glycan classification were performed using Genedata Expressionist via a specialised workflow that used libraries of previously detected glycopeptides to greatly reduce processing time. Two different spike proteins from six manufacturers were analysed. There was a strong similarity at each site across batches and manufacturers. The majority of the glycans present were of the truncated class, although at sites N61, N234, and N717/714 high mannose structures were dominant and at N1173/1170 aglycosylation was dominant for both variant proteins. A comparison was performed on a commercially available spike protein and our results were found to be similar to those of earlier reports. Our data clearly show that the overall glycosylation pattern of both spike protein variants was highly similar from batch to batch, and between materials produced at different manufacturing facilities. The use of our glycopeptide libraries greatly expedited the generation of site-specific glycan occupancy data for a large glycoprotein. We compared our method with previously obtained data from a commercially available insect cell-derived spike protein and the results were comparable to published findings.

Systems-Wide Site-Specific Analysis of Glycoproteins.

Glycosylation is one of the most common and complex post-translation modifications that influence the structural and functional properties of proteins. Glycoproteins are highly heterogeneous and exhibit site- and protein-specific expression differences. Mass spectrometry in combination with liquid chromatography has emerged as the most powerful tool for the comprehensive characterization of glycosylation. The analysis of intact glycopeptides has emerged as a promising strategy to analyze glycoproteins for their glycan heterogeneity at both protein- and site-specific levels. Nevertheless, intact glycopeptide characterization is challenging as elucidation of the glycan and peptide moieties requires specific sample preparation workflows that, combined with the tandem mass spectrometry approach, enable the identification of single glycopeptide species. In this chapter, we provide a detailed description of the methods that include procedures for (i) proteolytic digestion using specific proteases, (ii) optional glycopeptide enrichment using hydrophilic interaction liquid chromatography, (iii) nano-LC-MS/MS analysis of glycopeptides, and (iv) data analysis for identification of glycopeptides. Together, our workflow provides a framework for the system-wide site-specific analysis of N- and O-glycopeptides derived from complex biological or clinical samples.

Site-Specific Intact N-Linked Glycopeptide Characterization of Prostate-Specific Membrane Antigen from Metastatic Prostate Cancer Cells.

The composition of N-linked glycans that are conjugated to the prostate-specific membrane antigen (PSMA) and their functional significance in prostate cancer progression have not been fully characterized. PSMA was isolated from two metastatic prostate cancer cell lines, LNCaP and MDAPCa2b, which have different tissue tropism and localization. Isolated PSMA was trypsin-digested, and intact glycopeptides were subjected to LC-HCD-EThcD-MS/MS analysis on a Tribrid Orbitrap Fusion Lumos mass spectrometer. Differential qualitative and quantitative analysis of site-specific N-glycopeptides was performed using Byonic and Byologic software. Comparative quantitative analysis demonstrates that multiple glycopeptides at asparagine residues 51, 76, 121, 195, 336, 459, 476, and 638 were in significantly different abundance in the two cell lines (p < 0.05). Biochemical analysis using endoglycosidase treatment and lectin capture confirm the MS and site occupancy data. The data demonstrate the effectiveness of the strategy for comprehensive analysis of PSMA glycopeptides. This approach will form the basis of ongoing experiments to identify site-specific glycan changes in PSMA isolated from disease-stratified clinical samples to uncover targets that may be associated with disease progression and metastatic phenotypes.

Complementary proteome and glycoproteome access revealed through comparative analysis of reversed phase and porous graphitic carbon chromatography.

Continual developments in instrumental and analytical techniques have aided in establishing rigorous connections between protein glycosylation and human illness. These illnesses, such as various forms of cancer, are often associated with poor prognoses, prompting the need for more comprehensive characterization of the glycoproteome. While innovative instrumental and computational strategies have largely benefited glycoproteomic analyses, less attention is given to benefits gained through alternative, optimized chromatographic techniques. Porous graphitic carbon (PGC) chromatography has gained considerable interest in glycomics research due to its mobile phase flexibility, increased retention of polar analytes, and improved structural elucidation at higher temperatures. PGC has yet to be systematically compared against or in tandem with standard reversed phase liquid chromatography (RPLC) in high-throughput bottom-up glycoproteomic experiments, leaving the potential benefits unexplored. Performing comparative analysis of single and biphasic separation regimes at a range of column temperatures illustrates complementary advantages for each method. PGC separation is shown to selectively retain shorter, more hydrophilic glycopeptide species, imparting higher average charge, and exhibiting greater microheterogeneity coverage for identified glycosites. Additionally, we demonstrate that liquid-phase separation of glycopeptide isomers may be achieved through both single and biphasic PGC separations, providing a means towards facile, multidimensional glycopeptide characterization. Beyond this, we demonstrate how utilization of multiple separation regimes and column temperatures can aid in profiling the glycoproteome in tumorigenic and aggressive prostate cancer cells. RAW MS proteomic and glycoproteomic datasets have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD024196 (10.6019/PXD024196) and PXD024195, respectively.

Evaluating the Performance of 193 nm Ultraviolet Photodissociation for Tandem Mass Tag Labeled Peptides

Despite the successful application of tandem mass tags (TMT) for peptide quantitation, missing reporter ions in higher energy collisional dissociation (HCD) spectra remains a challenge for consistent quantitation, especially for peptides with labile post-translational modifications. Ultraviolet photodissociation (UVPD) is an alternative ion activation method shown to provide superior coverage for sequencing of peptides and intact proteins. Here, we optimized and evaluated 193 nm UVPD for the characterization of TMT-labeled model peptides, HeLa proteome, and N-glycopeptides from model proteins. UVPD yielded the same TMT reporter ions as HCD, at m/z 126–131. Additionally, UVPD produced a wide range of fragments that yielded more complete characterization of glycopeptides and less frequent missing TMT reporter ion channels, whereas HCD yielded a strong tradeoff between characterization and quantitation of TMT-labeled glycopeptides. However, the lower fragmentation efficiency of UVPD yielded fewer peptide identifications than HCD. Overall, 193 nm UVPD is a valuable tool that provides an alternative to HCD for the quantitation of large and highly modified peptides with labile PTMs. Continued development of instrumentation specific to UVPD will yield greater fragmentation efficiency and fulfil the potential of UVPD to be an all-in-one spectrum ion activation method for broad use in the field of proteomics.

Hollow MnFe2O4@C@APBA Nanospheres with Size Exclusion and pH Response for Efficient Enrichment of Endogenous Glycopeptides

Enrichment and detection of glycopeptides are an important clinical measure for the diagnosis of complex diseases. Enrichment materials play a key role in this process; they must have an effective sample-screening ability to eliminate the interference of nonglycopeptides. In this work, novel hollow MnFe2O4@C@APBA nanospheres (HMCAs) with magnetic and pH responsiveness were prepared for glycopeptide enrichment. The as-prepared composites have a suitable hollow structure and large specific surface area, and the boron hydroxyl group in their cavities can fix or disconnect the hydrophilic groups of the glycopeptides at different pH, so the glycopeptides can be adsorbed or desorbed in a controllable way. Enrichment results showed that the HMCAs exhibited an excellent enrichment performance: ultralow limit of detection (approximately 0.5 fmol μL-1), perfect size-exclusion effect (HRP/BSA, 1:800, w/w), favorable universality (HRP, IgG, and RNase B), and high binding capacity (150 mg/g). In order to verify the application of materials in practice, the HMCAs were used for the analysis of complex samples and it was found that 474 glycopeptides were identified from 210 glycoproteins in three replicate analyses of 2 μL of human serum. The results showed that the HMCAs could be used as a promising enrichment material for glycopeptide characterization in MS-based glycoproteomics and related fields.

Neutrophil myeloperoxidase harbors distinct site-specific peculiarities in its glycosylation

Anti-neutrophil cytoplasmic autoantibodies (ANCAs) are directed against lysosomal components of neutrophils. ANCAs directed to proteinase 3 and myeloperoxidase (MPO) in particular are associated with distinct forms of small vessel vasculitides. MPO is an abundant neutrophil-derived heme protein that is part of the antimicrobial defense system. The protein is typically present in the azurophilic granules of neutrophils, but a large portion may also enter the extracellular space. It remains unclear why MPO is frequently the target of antibody-mediated autoimmune responses. MPO is a homodimeric glycoprotein, posttranslationally modified with complex sugars at specific sites. Glycosylation can strongly influence protein function, affecting its folding, receptor interaction, and backbone accessibility. MPO potentially can be heavily modified as it harbors 5 putative N-glycosylation sites (10 in the mature dimer). Although considered important for MPO structure and function, the full scope and relative abundance of the glycans attached to MPO is unknown. Here, combining bottom-up glycoproteomics and native MS approaches, we structurally characterized MPO from neutrophils of healthy human donors. We quantified the relative occupancy levels of the glycans at each of the five sites and observed complex heterogeneity and site-specific glycosylation. In particular, we detected glycosylation phenotypes uncommon for glycoproteins in the extracellular space, such as a high abundance of phosphorylated high-mannose species and severely truncated small glycans having the size of paucimannose or smaller. We hypothesize that the atypical glycosylation pattern found on MPO might contribute to its specific processing and presentation as a self-antigen by antigen-presenting cells.

Glyco-DIA: a method for quantitative O-glycoproteomics with in silico-boosted glycopeptide libraries.

We report a liquid chromatography coupled to tandem mass spectrometry O-glycoproteomics strategy using data-independent acquisition (DIA) mode for direct analysis of O-glycoproteins. This approach enables characterization of glycopeptides and structures of O-glycans on a proteome-wide scale with quantification of stoichiometries (though it does not allow for direct unambiguous glycosite identification). The method relies on a spectral library of O-glycopeptides; the Glyco-DIA library contains sublibraries obtained from human cell lines and human serum, and it currently covers 2,076 O-glycoproteins (11,452 unique glycopeptide sequences) and the 5 most common core1 O-glycan structures. Applying the Glyco-DIA library to human serum without enrichment for glycopeptides enabled us to identify and quantify 269 distinct glycopeptide sequences bearing up to 5 different core1 O-glycans from 159 glycoproteins in a SingleShot analysis.

Characterization of intact glycopeptides reveals the impact of culture media on site-specific glycosylation of EPO-Fc fusion protein generated by CHO-GS cells.

With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N- and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N- and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc N-glycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi- and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1-4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N- and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest.

The Fine Art of Destruction: A Guide to In‐Depth Glycoproteomic Analyses—Exploiting the Diagnostic Potential of Fragment Ions

The unambiguous mass spectrometric identification and characterization of glycopeptides is crucial to elucidate the micro- and macroheterogeneity of glycoproteins. Here, combining lower and stepped collisional energy fragmentation for the in-depth and site-specific analysis of N- and O-glycopeptides is proposed. Using a set of four representative and biopharmaceutically relevant glycoproteins (IgG, fibrinogen, lactotransferrin, and ribonucleaseB), the benefits and limitations of the developed workflow are highlighted and a state-of-the-art blueprint for conducting high-quality in-depth N- and O-glycoproteomic analyses is provided. Further, a modified and improved version of cotton hydrophilic interaction liquid chromatography-based solid phase extraction for glycopeptide enrichment is described. For the unambiguous identification of N-glycopeptides, the use of a conserved yet, rarely employed-fragmentation signature [Mpeptide +H+0,2 XGlcNAc]+ is proposed. It is shown for the first time that this fragmentation signature can consistently be found across all N-glycopeptides, but not on O-glycopeptides. Moreover, the use of the relative abundance of oxonium ions to retrieve glycan structure information, for example, differentiation of hybrid- and high-mannose-type N-glycans or differentiation between antenna GlcNAc and bisecting GlcNAc, is systematically and comprehensively evaluated. The findings may increase confidence and comprehensiveness in manual and software-assisted glycoproteomics.

Characterization of glycopeptides using a stepped higher-energy C-trap dissociation approach on a hybrid quadrupole orbitrap.

Accurate characterization of glycopeptides without a prior glycan cleavage could provide valuable information on site-specific glycosylation, which is critical to reveal the biological functions of protein glycosylation. However, due to the distinct nature of oligosaccharides and ploypeptides, it is usually difficult to effectively fragment glycopeptides in mass spectrometry analysis. Here we applied a stepped normalized collisional energy (NCE) approach, which is able to combine fragment ions from three different collision energies, in a hybrid quadrupole orbitrap (Q Exactive Plus) to characterize glycopeptides. A systematic evaluation was firstly performed to find optimal NCE values for the fragmentation of glycan chains and peptide backbones from glycopeptides. Guided by the results of the systematic evaluation, the stepped NCE method was optimized and employed to analyze glycopeptides enriched from human serum. The stepped NCE approach was found to effectively fragment both the glycan chains and peptide backbones from glycopeptides and record these fragments in a single MS/MS spectrum. In comparison with the regular HCD methods, the stepped NCE method identified more glycopeptides with higher scores from human serum samples. Our studies demonstrate the capability of stepped NCE for the effective characterization of glycopeptides on a large scale.

Study of structure‐dependent chromatographic behavior of glycopeptides using reversed phase nanoLC

Analysis of glycosylation is challenging due to micro- and macro-heterogeneity of the protein attachment. A combination of LC with MS/MS is one of the most powerful tools for glycopeptide analysis. In this work, we show the effect of various monosaccharide units on the retention time of glycopeptides. Retention behavior of several glycoforms of six peptides obtained from tryptic digest of haptoglobin, hemopexin, and sex hormone-binding globulin was studied on a reversed phase chromatographic column. We observed reduction of the retention time with increasing number of monosaccharide units of glycans attached to the same peptide backbone. Fucosylation of larger glycans provides less significant retention time shift than for smaller ones. Retention times of glycopeptides were expressed as relative retention times. These relative retention times were used for calculation of upper and lower limits of glycopeptide retention time windows under the reversed phase conditions. We then demonstrated on the case of a glycopeptide of haptoglobin that the predicted retention time window boosts confidence of identification and minimizes false-positive identification. Relative retention time, as a qualitative parameter, is expected to improve LC-MS/MS characterization of glycopeptides.

Identification and Characterization of Glycopeptides from Egg Protein Ovomucin with Anti-Agglutinating Activity against Porcine K88 Enterotoxigenic Escherichia coli Strains.

Ovomucin is a glycoprotein from egg white with potential to act as an anti-adhesive agent against infectious diseases. This study aimed to determine whether ovomucin or ovomucin hydrolysates could prevent adhesion of two porcine K88 enterotoxigenic Escherichia coli (ETEC) strains. Adhesion was assessed in vitro using a hemagglutination assay. Ovomucin hydrolysates, but not intact ovomucin, prevented adhesion of ETEC to porcine erythrocytes. The ovomucin hydrolysate prepared by acid protease II exhibited the best anti-agglutinating activity against both strains; this hydrolysate was fractionated by cation exchange chromatography and reverse-phase high-performance liquid chromatography (HPLC). The most active fractions, F3(9) and F7(1), with minimal inhibitory concentrations of 0.03 and 0.25 g/L against strains ECL13795 and ECL13998, respectively, were subjected to structural characterization. Six identified glycopeptides were all derived from α-ovomucin, composed of a pentasaccharide core of two N-acetylglucosamine and three mannose residues (GlcNAc2Man3) and a bisecting N-acetylglucosamine (GlcNAc). The terminal β-linked galactose from these glycopeptides could be one of the binding sites for K88ac fimbriae.

Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS

Purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of glycopeptides, originating from protease digests of glycoproteins, enables site-specific analysis of protein N- and O-glycosylations. We have described a protocol to enrich, hydrolyze by chondroitinase ABC, and characterize chondroitin sulfate-containing glycopeptides (CS-glycopeptides) using positive mode LC-MS/MS. The CS-glycopeptides, originating from the Bikunin proteoglycan of human urine samples, had ΔHexAGalNAcGlcAGalGalXyl-O-Ser hexasaccharide structure and were further substituted with 0-3 sulfate and 0-1 phosphate groups. However, it was not possible to exactly pinpoint sulfate attachment residues, for protonated precursors, due to extensive fragmentation of sulfate groups using high-energy collision induced dissociation (HCD). To circumvent the well-recognized sulfate instability, we now introduced Na+ ions to form sodiated precursors, which protected sulfate groups from decomposition and facilitated the assignment of sulfate modifications. Sulfate groups were pinpointed to both Gal residues and to the GalNAc of the hexasaccharide structure. The intensities of protonated and sodiated saccharide oxonium ions were very prominent in the HCD-MS2 spectra, which provided complementary structural analysis of sulfate substituents of CS-glycopeptides. We have demonstrated a considerable heterogeneity of the bikunin CS linkage region. The realization of these structural variants should be beneficial in studies aimed at investigating the importance of the CS linkage region with regards to the biosynthesis of CS and potential interactions to CS binding proteins. Also, the combined use of protonated and sodiated precursors for positive mode HCD fragmentation analysis will likely become useful for additional classes of sulfated glycopeptides.Graphical ᅟ

Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS.

Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. We sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry (MS). Using alpha-1-acid glycoprotein (AGP) as a model N-glycoprotein, we identified its tryptic N-glycopeptides and examined the data reproducibility in seven laboratories running different LC-MS/MS platforms. We used three test samples and one blind sample to evaluate instrument performance with entire sample preparation workflow. 165 site-specific N-glycopeptides representative of all N-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on the MS analyzer. Orbitrap Elite identified the greatest number of AGP N-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycan-cleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Laboratory proficiency affected the number of identified N-glycopeptides. The relative quantities of the 10 major N-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Quantitative analysis showed that the coefficient of variation was <25% for all test samples. Our analytical protocol yielded identification and quantification of site-specific N-glycopeptide isoforms of AGP from control and disease plasma sample.

SweetNET: A Bioinformatics Workflow for Glycopeptide MS/MS Spectral Analysis.

Glycoproteomics has rapidly become an independent analytical platform bridging the fields of glycomics and proteomics to address site-specific protein glycosylation and its impact in biology. Current glycopeptide characterization relies on time-consuming manual interpretations and demands high levels of personal expertise. Efficient data interpretation constitutes one of the major challenges to be overcome before true high-throughput glycopeptide analysis can be achieved. The development of new glyco-related bioinformatics tools is thus of crucial importance to fulfill this goal. Here we present SweetNET: a data-oriented bioinformatics workflow for efficient analysis of hundreds of thousands of glycopeptide MS/MS-spectra. We have analyzed MS data sets from two separate glycopeptide enrichment protocols targeting sialylated glycopeptides and chondroitin sulfate linkage region glycopeptides, respectively. Molecular networking was performed to organize the glycopeptide MS/MS data based on spectral similarities. The combination of spectral clustering, oxonium ion intensity profiles, and precursor ion m/z shift distributions provided typical signatures for the initial assignment of different N-, O- and CS-glycopeptide classes and their respective glycoforms. These signatures were further used to guide database searches leading to the identification and validation of a large number of glycopeptide variants including novel deoxyhexose (fucose) modifications in the linkage region of chondroitin sulfate proteoglycans.