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Bacterial pathogens can cause a broad range of infections with detrimental effects on health. Vaccine development is essential as multi-drug resistance in bacterial infections is a rising concern. Recombinantly produced proteins carrying O-antigen glycosylation are promising glycoconjugate vaccine candidates to prevent bacterial infections. However, methods for their comprehensive structural characterization are lacking. Here, we present a bottom-up approach for their site-specific characterization, detecting N-glycopeptides by nano reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS). Glycopeptide analyses revealed information on partial site-occupancy and site-specific glycosylation heterogeneity and helped corroborate the polysaccharide structures and their modifications. Bottom-up analysis was complemented by intact glycoprotein analysis using nano RP-LC-MS allowing the fast visualization of the polysaccharide distribution in the intact glycoconjugate. At the glycopeptide level, the model glycoconjugates analyzed showed different repeat unit (RU) distributions that spanned from 1 to 21 RUs attached to each of the different glycosylation sites. Interestingly, the intact glycoprotein analysis displayed a RU distribution ranging from 1 to 28 RUs, showing the predominant species when the different glycopeptide distributions are combined in the intact glycoconjugate. The complete workflow based on LC-MS measurements allows detailed and comprehensive analysis of the glycosylation state of glycoconjugate vaccines.

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BACKGROUND: Drosophila melanogaster is a well-studied and highly tractable genetic model system for deciphering the molecular mechanisms underlying various biological processes. Although being one of the most critical post-translational modifications of proteins, the understanding of glycosylation in Drosophila is still lagging behind compared with that of other model organisms. METHODS: In this study, we systematically investigated the site-specific N-glycan profile of Drosophila melanogaster using intact glycopeptide analysis technique. This approach identified the glycans, proteins, and their glycosites in Drosophila, as well as information on site-specific glycosylation, which allowed us to know which glycans are attached to which glycosylation sites. RESULTS: The results showed that the majority of N-glycans in Drosophila were high-mannose type (69.3%), consistent with reports in other insects. Meanwhile, fucosylated N-glycans were also highly abundant (22.7%), and the majority of them were mono-fucosylated. In addition, 24 different sialylated glycans attached with 16 glycoproteins were identified, and these proteins were mainly associated with developmental processes. Gene ontology analysis showed that N-glycosylated proteins in Drosophila were involved in multiple biological processes, such as axon guidance, N-linked glycosylation, cell migration, cell spreading, and tissue development. Interestingly, we found that seven glycosyltransferases and four glycosidases were N-glycosylated, which suggested that N-glycans may play a regulatory role in the synthesis and degradation of N-glycans and glycoproteins. CONCLUSIONS: To our knowledge, this work represents the first comprehensive analysis of site-specific N-glycosylation in Drosophila, thereby providing new perspectives for the understanding of biological functions of glycosylation in insects.

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Mass spectrometry-based discovery glycoproteomics is highly dependent on the use of chromatography paradigms amenable to analyte retention and separation. When compared against established stationary phases such as reversed-phase and hydrophilic interaction liquid chromatography, reports utilizing porous graphitic carbon have detailed its numerous advantages. Recent efforts have highlighted the utility in porous graphitic carbon in high-throughput glycoproteomics, principally through enhanced profiling depth and liquid-phase resolution at higher column temperatures. However, increasing column temperature has been shown to impart disparaging effects in glycopeptide identification. Herein we further elucidate this trend, describing qualitative and semiquantitative effects of increased column temperature on glycopeptide identification rates, signal intensity, resolution, and spectral count linear response. Through analysis of enriched bovine and human glycopeptides, species with high mannose and sialylated glycans were shown to most significantly benefit and suffer from high column temperatures, respectively. These results provide insight as to how porous graphitic carbon separations may be appropriately leveraged for glycopeptide identification while raising concerns over quantitative and semiquantitative label-free comparisons as the temperature changes. RAW MS glycoproteomic data are available via ProteomeXchange with identifier PXD034354.

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Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) is a powerful tool for the characterization and identification of the macro- and microheterogeneity of a glycoprotein in a bottom-up approach. This chapter describes in detail the sample preparation procedures using a purified biological sample, prostate-specific antigen, as a model protein, including proteolytic digestion (trypsin). In addition, insights are provided into the strengths of using capillary electrophoresis for obtaining isomer separation of differently linked sialic acids. Lastly, approaches and potential pitfalls for the integration and quantitation of glycopeptide signals from the obtained CZE-MS data are discussed.

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Age-related macular degeneration (AMD) has been associated with protective genetic variants in the ß1-3 glucosyltransferase (B3GLCT) locus through genome-wide association studies. B3GLCT mediates modification of proteins with thrombospondin type I repeats (TSR) that contain O-linked glucose ß1-3 fucose and C-linked mannose glycosylation motifs. B3GLCT-mediated modification is required for proper secretion of TSR-containing proteins. We aimed to start understanding the role of B3GLCT in AMD by evaluating its effect on glycosylation and secretion of proteins from retinal pigment epithelium (RPE) cells. We generated B3GLCT knockout (KO) RPE cells and analyzed glycosylation and secretion of thrombospondin 1 (TSP1), a protein involved in cellular processes highly relevant to AMD. Glycopeptide analysis confirmed the presence of the glucose-ß1,3-fucose product of B3GLCT on TSP1 in wildtype (WT) cells and its absence in KO cells. C-mannosylation was variably present on WT TSP1 and increased on TSR domains 1 and 3 in KO cells. Secretion of TSP1 was not affected by the absence of B3GLCT, even not when TSP1 was upregulated by TNFα treatment or when TSP1 was overexpressed in HEK293T cells. Future research is needed to elucidate the effect of the observed glycosylation defects in the context of AMD, which might involve functional loss of TSP1 or effects on other TSR proteins.

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Intact glycopeptide identification has long been known as a key and challenging barrier to the comprehensive and accurate understanding the role of glycosylation in an organism. Intact glycopeptide analysis is a blossoming field that has received increasing attention in recent years. MS-based strategies and relative software tools are major drivers that have greatly facilitated the analysis of intact glycopeptides, particularly intact N-glycopeptides. This article provides a systematic review of the intact glycopeptide-identification process using MS data generated in shotgun proteomic experiments, which typically focus on N-glycopeptide analysis. Particular attention is paid to the software tools that have been recently developed in the last decade for the interpretation and quality control of glycopeptide spectra acquired using different MS strategies. The review also provides information about the characteristics and applications of these software tools, discusses their advantages and disadvantages, and concludes with a discussion of outstanding tools.

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Early detection of prostate cancer may lead to the overdiagnosis and overtreatment of patients as well as missing significant cancers. The current diagnostic approach uses elevated serum concentrations of prostate-specific antigen (PSA) as an indicator of risk. However, this test has been widely criticized as it shows poor specificity and sensitivity. In order to improve early detection and diagnosis, several studies have investigated whether different PSA proteoforms are correlated to prostate cancer. Until now, studies and methodologies for the comprehensive characterization of PSA proteoforms from biofluids are scarce. For this purpose, we developed an intact protein assay to analyze PSA by capillary electrophoresis-electrospray ionization-mass spectrometry after affinity purification from patients' urine. Here, we determined six proteolytic cleavage variants. In regard to glycosylation, tri-, di-, mono- and non-sialylated complex-type N-glycans were found on non-cleaved PSA, as well as the non-glycosylated variant. The performance of the intact protein assay was assessed using a pooled sample, obtaining an inter-day variability of 15%. Furthermore, urinary patient samples were analyzed by intact protein analysis and a bottom-up approach (glycopeptide analysis). This combined approach revealed complimentary information on both levels, demonstrating the benefit of using two orthogonal techniques to provide a thorough profile of urinary PSA. SIGNIFICANCE: The detection of clinically relevant prostate cancer requires a more specific and sensitive biomarker and, in this case, several PSA proteoforms may be able to aid or improve the current PSA test. However, a comprehensive analysis of the intact PSA proteoform profile is still lacking. This study investigated the PSA proteoforms present in urine and, in particular, determined the relative contribution of cleaved PSA and non-cleaved PSA forms to the total glycosylation profile. Importantly, intact protein analysis did not require further sample treatment before being measured by CE-ESI-MS. Furthermore, its glycosylation was also assessed in a bottom-up approach to provide complementary information. Overall, these results represent an important basis for future characterization and biomarker studies.

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Many biotherapeutics such as monoclonal antibodies (mAb) and Fc-domain fusion proteins contain heterogeneous glycan contents at one or multiple glycosylation site(s). Site-specific glycan profile characterization is critical for monitoring the quality of these molecules during different stages of drug development. Hydrophilic interaction chromatography (HILIC) as an orthogonal separation method to reversed-phase liquid chromatography (RPLC) can achieve better glycopeptide identification due to the effective separation between individual glycoforms as well as the separation of glycopeptides from high-abundance non-glycosylated peptides, which can be further improved by modifying the mobile phases with ion-pairing agents (IP-HILIC). However, an online IP-HILIC coupled to mass spectrometry (MS) detection may suffer from the suppression of mass spectrometry signal during electrospray ionization due to the trifluoroacetic acid (TFA), commonly used as an ion-pairing agent. Here, we reported an optimized experimental condition for IP-HILIC-MS where glycine is added in the TFA-containing mobile phases to enhance the MS detection sensitivity for glycopeptides up to ~ 50-fold by eliminating the ion-suppression effect of an ion-pairing agent while still retaining excellent separation capacity. We demonstrated that with enhanced detection sensitivity, IP-HILIC-MS can confidently identify an increased number of site-specific N-linked glycans for IgG1, and IgG4 mAbs as well as an Fc-domain fusion protein (containing five N-glycosylation sites) through MS/MS-based search in the data-dependent acquisition mode, meanwhile, achieve comparable quantitative results compared with the traditional methods. We also demonstrated that IP-HILIC-MS can be used to identify low-level O-glycosylation and non-consensus N-glycosylation on mAbs without any enrichment prior to LC-MS analysis. Graphical abstract.

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With the size of the biopharmaceutical market exponentially increasing, there is an aligned growth in the importance of data-rich analyses, not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships. In monoclonal antibodies, many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known. The importance of their function focuses analytical research efforts on the development of robust, accurate and fast methods to support drug development and quality control. Released N-glycan analysis is considered as the gold standard for glycosylation characterisation; however, it is not the only method for quantitative analysis of glycoform heterogeneity. In this study, ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies. While observing good comparability between the quantitative results generated, it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.

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With the size of the biopharmaceutical market exponentially increasing, there is an aligned growth in the importance of data-rich analyses, not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships. In monoclonal antibodies, many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known. The importance of their function focuses analytical research efforts on the development of robust, accurate and fast methods to support drug development and quality control. Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however, it is not the only method for quantitative analysis of glycoform heterogeneity. In this study, ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies. While observing good comparability between the quantitative results generated, it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow ac-cording to application and the desired depth of data generated.

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Aberrant glycosylation is a hallmark of cancer that contributes to the disease's ability to evade the immune system. As the MHC processing pathways communicate cellular health to circulating CD8+ and CD4+ T-cells, MHC-associated glycopeptides are likely a source of neoantigens in cancer. In fact, recent advances in mass spectrometry have allowed for the detection and sequencing of tumor-specific glycopeptides from the MHC class I and class II processing pathways. Here, we describe methods for detecting, sequencing, and modeling these MHC-associated glycopeptides.

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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.

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Significant changes of glycan structures are observed in humans if diseases like cancer, arthritis or inflammation are present. Thus, interest in biomarkers based on glycan structures has rapidly emerged in recent years and monitoring disease specific changes of glycosylation and their quantification is of great interest. Mass spectrometry is most commonly used to characterize and quantify glycopeptides and glycans liberated from the glycoprotein of interest. However, ionization properties of glycopeptides can strongly depend on their composition and can therefore lead to intensities that do not reflect the actual proportions present in the intact glycoprotein. Here we show that an increase in the length of the peptide can lead to a more accurate determination and quantification of the glycans. The four glycosylation sites of human serum ceruloplasmin from 17 different individuals were analyzed using glycopeptides of varying peptide lengths, obtained by action of different proteases and by limited digestion. In most cases, highly sialylated compositions showed an increased relative abundance with increasing peptide length. We observed a relative increase of triantennary glycans of up to a factor of three and, even more, MS peaks corresponding to tetraantennary compositions on ceruloplasmin at glycosite 137N in all 17 samples, which we did not detect using a bottom up approach. The data presented here leads to the conclusion that a middle down - or when possible a top down - approach is favorable for qualitative and quantitative analysis of the glycosylation of glycoproteins.

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Sodium butyrate (NaBu) is not only well-known for enhancing protein production, but also degrades glycan quality. In this study, butyrate supplied by the precursor molecule 1,3,4-O-Bu3 ManNAc is applied to overcome the negative effects of NaBu on glycan quality while simultaneously increasing the productivity of the model recombinant erythropoietin (EPO). The beneficial impact of 1,3,4-O-Bu3 ManNAc on EPO glycan quality, while evident in wild-type CHO cells, is particularly pronounced in glycoengineered CHO cells with stable overexpression of ß-1,4- and ß-1,6-N-acetylglucosaminyltransferases (GnTIV and GnTV) and α-2,6-sialyltransferase (ST6) enzymes responsible for N-glycan antennarity and sialylation. Supplementation of 1,3,4-O-Bu3 ManNAc achieves approximately 30% sialylation enhancement on EPO protein in wild-type CHO cells. Overexpression of GnTIV/GnTV/ST6 in CHO cells increases EPO sialylation about 40%. Combining 1,3,4-O-Bu3 ManNAc treatment in glyocengineered CHO cells promotes EPO sialylation about 75% relative to EPO from wild-type CHO cells. Moreover, a detailed mass spectrometric ESI-LC-MS/MS characterization of glycans at each of the three N-glycosylation sites of EPO showed that the 1st N-site is highly sialylated and either the negative impact of NaBu or the beneficial effect 1,3,4-O-Bu3 ManNAc treatments mainly affects the 2nd and 3rd N-glycan sites of EPO protein. In summary, these results demonstrate 1,3,4-O-Bu3 ManNAc can compensate for the negative effect of NaBu on EPO glycan quality while simultaneously enhancing recombinant protein yields. In this way, a platform that integrates glycoengineering with metabolic supplementation can result in synergistic improvements in both production and glycosylation in CHO cells.

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The MHC class II (MHCII) processing pathway presents peptides derived from exogenous or membrane-bound proteins to CD4+ T cells. Several studies have shown that glycopeptides are necessary to modulate CD4+ T cell recognition, though glycopeptide structures in these cases are generally unknown. Here, we present a total of 93 glycopeptides from three melanoma cell lines and one matched EBV-transformed line with most found only in the melanoma cell lines. The glycosylation we detected was diverse and comprised 17 different glycoforms. We then used molecular modeling to demonstrate that complex glycopeptides are capable of binding the MHC and may interact with complementarity determining regions. Finally, we present the first evidence of disulfide-bonded peptides presented by MHCII. This is the first large scale study to sequence glyco- and disulfide bonded MHCII peptides from the surface of cancer cells and could represent a novel avenue of tumor activation and/or immunoevasion.

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Protein glycosylation drives many biological processes and serves as markers for disease; therefore, the development of tools to study glycosylation is an essential and growing area of research. Mass spectrometry can be used to identify both the glycans of interest and the glycosylation sites to which those glycans are attached, when proteins are proteolytically digested and their glycopeptides are analyzed by a combination of high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) methods. One major challenge in these experiments is collecting the requisite MS/MS data. The digested glycopeptides are often present in complex mixtures and in low abundance, and the most commonly used approach to collect MS/MS data on these species is data-dependent acquisition (DDA), where only the most intense precursor ions trigger MS/MS. DDA results in limited glycopeptide coverage. Semi-targeted data acquisition is an alternative experimental approach that can alleviate this difficulty. However, due to the massive heterogeneity of glycopeptides, it is not obvious how to expediently generate inclusion lists for these types of analyses. To solve this problem, we developed the software tool GlycoPep MassList, which can be used to generate inclusion lists for liquid chromatography tandem-mass spectrometry (LC-MS/MS) experiments. The utility of the software was tested by conducting comparisons between semi-targeted and untargeted data-dependent analysis experiments on a variety of proteins, including IgG, a protein whose glycosylation must be characterized during its production as a biotherapeutic. When the GlycoPep MassList software was used to generate inclusion lists for LC-MS/MS experiments, more unique glycopeptides were selected for fragmentation. Generally, ∼30 % more unique glycopeptides can be analyzed per protein, in the simplest cases, with low background. In cases where background ions from proteins or other interferents are high, usage of an inclusion list is even more advantageous. The software is freely publically accessible. Graphical abstract Software increases the number of glycopeptides that get selected for MS/MS analysis.

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This chapter contains a nanoscale liquid chromatography-mass spectrometry method for the glycoform profiling of the conserved Fc N-glycosylation site of monoclonal and polyclonal immunoglobulin G (IgG). It describes in detail LaCyTools, a program for automated data (pre-)processing of the obtained LC-MS data. The minimal sample preparation necessary is explained as well as an optional method for affinity purification of (polyclonal) antibodies from serum or plasma.After (optional) affinity purification, the pure IgG is cleaved with trypsin. The tryptic glycopeptides are separated almost exclusively on their peptide backbone. This ensures similar response factors for all glycoforms in the MS detection and allows the collection of separate glycoform profiles for different IgG isoforms or allotypes. LaCyTools automatically performs label-free (relative) quantitation of the obtained data after minimal manual input and additionally calculates several quality criteria which can be used for data curation at the level of both individual analytes and entire LC-MS runs.

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Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder caused by mutations in the Pkd1 or Pkd2 genes, in which large cysts replace normal kidney tissue, leading to end-stage kidney disease. In this study we have utilized a powerful nano-HPLC-mass spectrometric approach to characterize patterns of normal and abnormal N-linked glycosylation of α3 integrin subunit in Pkd1(-/-) cells derived from mouse kidneys. Higher molecular weight glycan structures with a different monosaccharide composition were observed at two sites, namely, Asn-925 and Asn-928 sites in α3 integrin isolated from Pkd1(+/+) cells compared with Pkd1(-/-) cells. In addition, an unusual and unique disialic acid glycan structure was observed solely in Pkd1(-/-) cells. Thus, these studies suggest that abnormal protein glycosylation may have a role on the pathogenesis of cyst formation in ADPKD.

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