(Sialyl)Lewis Antigen Expression on Glycosphingolipids, N-, and O-Glycans in Colorectal Cancer Cell Lines is Linked to a Colon-Like Differentiation Program.

Alterations in the glycomic profile are a hallmark of cancer, including colorectal cancer (CRC). While, the glycosylation of glycoproteins and glycolipids has been widely studied for CRC cell lines and tissues, a comprehensive overview of CRC glycomics is still lacking due to the usage of different samples and analytical methods. In this study, we compared glycosylation features of N-, O-glycans, and glycosphingolipid glycans for a set of 22 CRC cell lines, all measured by porous graphitized carbon nano-liquid chromatography-tandem mass spectrometry. An overall, high abundance of (sialyl)Lewis antigens for colon-like cell lines was found, while undifferentiated cell lines showed high expression of H blood group antigens and α2-3/6 sialylation. Moreover, significant associations of glycosylation features were found between the three classes of glycans, such as (sialyl)Lewis and H blood group antigens. Integration of the datasets with transcriptomics data revealed positive correlations between (sialyl)Lewis antigens, the corresponding glycosyltransferase FUT3 and transcription factors CDX1, ETS, HNF1/4A, MECOM, and MYB. This indicates a possible role of these transcription factors in the upregulation of (sialyl)Lewis antigens, particularly on glycosphingolipid glycans, via FUT3/4 expression in colon-like cell lines. In conclusion, our study provides insights into the possible regulation of glycans in CRC and can serve as a guide for the development of diagnostic and therapeutic biomarkers.

High-Throughput Glycomic Methods.

Glycomics aims to identify the structure and function of the glycome, the complete set of oligosaccharides (glycans), produced in a given cell or organism, as well as to identify genes and other factors that govern glycosylation. This challenging endeavor requires highly robust, sensitive, and potentially automatable analytical technologies for the analysis of hundreds or thousands of glycomes in a timely manner (termed high-throughput glycomics). This review provides a historic overview as well as highlights recent developments and challenges of glycomic profiling by the most prominent high-throughput glycomic approaches, with N-glycosylation analysis as the focal point. It describes the current state-of-the-art regarding levels of characterization and most widely used technologies, selected applications of high-throughput glycomics in deciphering glycosylation process in healthy and disease states, as well as future perspectives.

High Diversity of Glycosphingolipid Glycans of Colorectal Cancer Cell Lines Reflects the Cellular Differentiation Phenotype.

Colorectal cancer (CRC)-associated changes of protein glycosylation have been widely studied. In contrast, the expression of glycosphingolipid (GSL) patterns in CRC has, hitherto, remained largely unexplored. Even though GSLs are major carriers of cell surface carbohydrates, they are understudied due to their complexity and analytical challenges. In this study, we provide an in-depth analysis of GSL glycans of 22 CRC cell lines using porous graphitized carbon nano-liquid chromatography coupled with electrospray ionization-mass spectrometry. Our data revealed that the GSL expression varies among different cell line classifications, with undifferentiated cell lines showing high expression of blood group A, B, and H antigens while for colon-like cell lines the most prominent GSL glycans contained (sialyl)-LewisA/X and LewisB/Y antigens. Moreover, the GSL expression correlated with relevant glycosyltransferases that are involved in their biosynthesis as well as with transcription factors (TFs) implicated in colon differentiation. Additionally, correlations between certain glycosyltransferases and TFs at mRNA expression level were found, such as FUT3, which correlated with CDX1, ETS2, HNF1A, HNF4A, MECOM, and MYB. These TFs are upregulated in colon-like cell lines pointing to their potential role in regulating fucosylation during colon differentiation. In conclusion, our study reveals novel layers of potential GSL glycans regulation relevant for future research in colon differentiation and CRC.

Software-Assisted Data Processing Workflow for Intact Glycoprotein Mass Spectrometry.

Intact protein analysis by mass spectrometry is important for several applications such as assessing post-translational modifications and biotransformation. In particular, intact protein analysis allows the detection of proteoforms that are commonly missed by other approaches such as proteolytic digestion followed by bottom-up analysis. Two quantification methods are mainly used for intact protein data quantification, namely the extracted ion and deconvolution approaches. However, a consensus with regard to a single best practice for intact protein data processing is lacking. Furthermore, many data processing tools are not fit-for-purpose and, as a result, the analysis of intact proteins is laborious and lacks the throughput required to be implemented for the analysis of clinical cohorts. Therefore, in this study, we investigated the application of a software-assisted data analysis and processing workflow in order to streamline intact protein integration, annotation, and quantification via deconvolution. In addition, the assessment of orthogonal data sets generated via middle-up and bottom-up analysis enabled the cross-validation of cleavage proteoform assignments present in seminal prostate-specific antigen (PSA). Furthermore, deconvolution quantification of PSA from patients' urine revealed results that were comparable with manually performed quantification based on extracted ion electropherograms. Overall, the presented workflow allows fast and efficient processing of intact protein data. The raw data is available on MassIVE using the identifier MSV000086699.

High sensitivity glycomics in biomedicine.

Many analytical challenges in biomedicine arise from the generally high heterogeneity and complexity of glycan- and glycoconjugate-containing samples, which are often only available in minute amounts. Therefore, highly sensitive workflows and detection methods are required. In this review mass spectrometric workflows and detection methods are evaluated for glycans and glycoproteins. Furthermore, glycomic methodologies and innovations that are tailored for enzymatic treatments, chemical derivatization, purification, separation, and detection at high sensitivity are highlighted. The discussion is focused on the analysis of mammalian N-linked and GalNAc-type O-linked glycans.

In-Depth Analysis of the N-Glycome of Colorectal Cancer Cell Lines.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer deaths worldwide. A well-known hallmark of cancer is altered glycosylation. Analyzing the N-glycosylation of CRC cell lines may provide potential therapeutic or diagnostic targets. In this study, an in-depth N-glycomic analysis of 25 CRC cell lines was conducted using porous graphitized carbon nano-liquid chromatography coupled to electrospray ionization mass spectrometry. This method allows for the separation of isomers and performs structural characterization, revealing profound N-glycomic diversity among the studied CRC cell lines with the elucidation of a number of 139 N-glycans. A high degree of similarity between the two N-glycan datasets measured on the two different platforms (porous graphitized carbon nano-liquid chromatography electrospray ionization tandem mass spectrometry (PGC-nano-LC-ESI-MS) and matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS)) was discovered. Furthermore, we studied the associations between glycosylation features, glycosyltransferases (GTs), and transcription factors (TFs). While no significant correlations between the glycosylation features and GTs were found, the association between TF CDX1 and (s)Le antigen expression and relevant GTs FUT3/6 suggests that CDX1 contributes to the expression of the (s)Le antigen through the regulation of FUT3/6. Our study provides a comprehensive characterization of the N-glycome of CRC cell lines, which may contribute to the future discovery of novel glyco-biomarkers of CRC.

Glycosphingolipid-Glycan Signatures of Acute Myeloid Leukemia Cell Lines Reflect Hematopoietic Differentiation.

Aberrant expression of certain glycosphingolipids (GSLs) is associated with the differentiation of acute myeloid leukemia (AML) cells. However, the expression patterns of GSLs in AML are still poorly explored because of their complexity, the presence of multiple isomeric structures, and tedious analytical procedures. In this study, we performed an in-depth GSL glycan analysis of 19 AML cell lines using porous graphitized carbon liquid chromatography-mass spectrometry revealing strikingly different GSL glycan profiles between the various AML cell lines. The cell lines of the M6 subtype showed a high expression of gangliosides with α2,3-sialylation and Neu5Gc, while the M2 and M5 subtypes were characterized by high expression of (neo)lacto-series glycans and Lewis A/X antigens. Integrated analysis of glycomics and available transcriptomics data revealed the association of GSL glycan abundances with the transcriptomics expression of certain glycosyltransferases (GTs) and transcription factors (TFs). In addition, correlations were found between specific GTs and TFs. Our data reveal TFs GATA2, GATA1, and RUNX1 as candidate inducers of the expression of gangliosides and sialylation via regulation of the GTs ST3GAL2 and ST8SIA1. In conclusion, we show that GSL glycan expression levels are associated with hematopoietic AML classifications and TF and GT gene expression. Further research is needed to dissect the regulation of GSL expression and its role in hematopoiesis and associated malignancies.

The minimum information required for a glycomics experiment (MIRAGE): reporting guidelines for capillary electrophoresis.

The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative to standardize the reporting of glycoanalytical methods and to assess their reproducibility. To date, the MIRAGE Commission has published several reporting guidelines that describe what information should be provided for sample preparation methods, mass spectrometry methods, liquid chromatography analysis, exoglycosidase digestions, glycan microarray methods, and nuclear magnetic resonance methods. Here, we present the first version of reporting guidelines for glyco(proteo)mics analysis by capillary electrophoresis (CE) for standardized and high-quality reporting of experimental conditions in the scientific literature. The guidelines cover all aspects of a glyco(proteo)mics CE experiment including sample preparation, CE operation mode (CZE, CGE, CEC, MEKC, cIEF, cITP), instrument configuration, capillary separation conditions, detection, data analysis, and experimental descriptors. These guidelines are linked to other MIRAGE guidelines and are freely available through the project website https://www.beilstein-institut.de/en/projects/mirage/guidelines/#ce_analysis (doi:10.3762/mirage.7).

Sialic Acid Derivatization of Fluorescently Labeled N-Glycans Allows Linkage Differentiation by Reversed-Phase Liquid Chromatography-Fluorescence Detection-Mass Spectrometry.

Sialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of these monosaccharides are owed, in part, to linkage variants, and as a result, linkage-specific analysis of sialic acids is an important aspect of glycomic studies. This has been addressed by derivatization strategies using matrix-assisted laser desorption/ionization mass spectrometry (MS) or sialidase digestion arrays followed by liquid chromatography (LC)-MS. Despite this, these approaches are unable to simultaneously provide unambiguous assignment of sialic acid linkages and assess further isomeric glycan features within a single measurement. Thus, for the first time, we present the combination of procainamide fluorescent labeling with sialic acid linkage-specific derivatization via ethyl esterification and amidation for the analysis of released plasma N-glycans using reversed-phase (RP)LC-fluorescence detection (FD)-MS. As a result, α2,3- and α2,6-sialylated N-glycans, with the same mass prior to derivatization, are differentiated based on retention time, precursor mass, and fragmentation spectra, and additional sialylated isomers were also separated. Furthermore, improved glycan coverage and protocol precision were found via the novel application using a combined FD-MS quantification approach. Overall, this platform achieved unambiguous assignment of N-glycan sialic acid linkages within a single RPLC-FD-MS measurement, and by improving their retention on RPLC, this technique can be used for future investigations of released N-glycans as an additional or orthogonal method to current analytical approaches.

N-Glycan Isomer Differentiation by Zero Flow Capillary Electrophoresis Coupled to Mass Spectrometry.

Isomeric N-glycans often vastly differ in their biological activities, hence the need for methods that allow resolving and structurally characterizing them in biological material. Here, we established a zero flow approach using capillary electrophoresis in combination with (tandem) mass spectrometry to allow structural characterization of isomeric N-glycans at high sensitivity. Additionally, diagnostic fragment ion ratios were identified, indicative for the antenna carrying specifically linked sialic acids. In total, 208 N-glycans were characterized in human plasma, with 57 compositions showing multiple isomers.

Colorectal cancer cell lines show striking diversity of their O-glycome reflecting the cellular differentiation phenotype.

Alterations in protein glycosylation in colorectal cancer (CRC) have been extensively studied using cell lines as models. However, little is known about their O-glycome and the differences in glycan biosynthesis in different cell types. To provide a better understanding of the variation in O-glycosylation phenotypes and their association with other molecular features, an in-depth O-glycosylation analysis of 26 different CRC cell lines was performed. The released O-glycans were analysed on porous graphitized carbon nano-liquid chromatography system coupled to a mass spectrometer via electrospray ionization (PGC-nano-LC-ESI-MS/MS) allowing isomeric separation as well as in-depth structural characterization. Associations between the observed glycan phenotypes with previously reported cell line transcriptome signatures were examined by canonical correlation analysis. Striking differences are observed between the O-glycomes of 26 CRC cell lines. Unsupervized principal component analysis reveals a separation between well-differentiated colon-like and undifferentiated cell lines. Colon-like cell lines are characterized by a prevalence of I-branched and sialyl Lewis x/a epitope carrying glycans, while most undifferentiated cell lines show absence of Lewis epitope expression resulting in dominance of truncated α2,6-core sialylated glycans. Moreover, the expression of glycan signatures associates with the expression of glycosyltransferases that are involved in their biosynthesis, providing a deeper insight into the regulation of glycan biosynthesis in different cell types. This untargeted in-depth screening of cell line O-glycomes paves the way for future studies exploring the role of glycosylation in CRC development and drug response leading to discovery of novel targets for the development of anti-cancer antibodies.

Site-Specific N-Linked Glycosylation Analysis of Human Carcinoembryonic Antigen by Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry.

With 28 potential N-glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of N-linked glycosylation, and approximately 60% of its molecular mass can be attributed to its carbohydrates. CEA is often overexpressed and released by many solid tumors, including colorectal carcinomas. CEA displays an impressive heterogeneity and variability in sugar content; however, site-specific distribution of carbohydrate structures has not been reported so far. The present study investigated CEA samples purified from human colon carcinoma and human liver metastases and enabled the characterization of 21 out of 28 potential N-glycosylation sites with respect to their occupancy. The coverage was achieved by a multienzymatic digestion approach with specific enzymes, such as trypsin, endoproteinase Glu-C, and the nonspecific enzyme, Pronase, followed by analysis using sheathless CE-MS/MS. In total, 893 different N-glycopeptides and 128 unique N-glycan compositions were identified. Overall, a great heterogeneity was found both within (micro) and in between (macro) individual N-glycosylation sites. Moreover, notable differences were found on certain N-glycosylation sites between primary adenocarcinoma and metastatic tumor in regard to branching, bisection, sialylation, and fucosylation. Those features, if further investigated in a targeted manner, may pave the way toward improved diagnostics and monitoring of colorectal cancer progression and recurrence. Raw mass spectrometric data and Skyline processed data files that support the findings of this study are available in the MassIVE repository with the identifier MSV000086774 [DOI: 10.25345/C5Z50X].

Dopant-Enriched Nitrogen Gas for Enhanced Electrospray Ionization of Released Glycans in Negative Ion Mode.

The desolvation and ionization process of analytes can significantly be improved by enriching the nebulizing gas with a dopant (dopant enriched nitrogen (DEN) gas) in the electrospray source. However, for the analysis of released glycans in negative ion mode, the usage of DEN gas remains largely unexplored. For this purpose, we investigated the effect of different polar protic solvents (methanol, ethanol, and isopropanol) as well as using solely the nebulizing gas or ambient air on the ionization and charge state distribution of released N- and O-glycans. Compared to the standard acetonitrile enriched nitrogen gas, isopropanol showed the highest increase in regards to peak areas. Moreover, it showed large benefits for the identification of glycan structures at high sensitivity as the increased precursor intensities subsequently resulted in higher intensities in tandem MS mode. While similar effects are noted for both neutral and sialylated species, the most significant effect was observed for early eluting glycans where very low acetonitrile concentrations were present in the eluent. The best results in terms of S/N ratios were obtained with methanol, with less effect on the MS/MS signal enhancement. Therefore, the use of this dopant would be particularly beneficial for high sensitivity MS-mode applications. In conclusion, isopropanol enriched DEN gas greatly improves the detection of both N-and O-glycan species and their tandem mass spectra, particularly for the early eluting species whose ionization in the absence of DEN gas is hindered by low organic concentrations.

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

Plasma N-Glycan Signatures Are Associated With Features of Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Biomarkers are needed for early detection of Crohn's disease (CD) and ulcerative colitis (UC) or to predict patient outcomes. Glycosylation is a common and complex posttranslational modification of proteins that affects their structure and activity. We compared plasma N-glycosylation profiles between patients with CD or UC and healthy individuals (controls). METHODS: We analyzed the total plasma N-glycomes of 2635 patients with inflammatory bowel diseases and 996 controls by mass spectrometry with a linkage-specific sialic acid derivatization technique. Plasma samples were acquired from 2 hospitals in Italy (discovery cohort, 1989 patients with inflammatory bowel disease [IBD] and 570 controls) and 1 medical center in the United States (validation cohort, 646 cases of IBD and 426 controls). Sixty-three glycoforms met our criteria for relative quantification and were extracted from the raw data with the software MassyTools. Common features shared by the glycan compositions were combined in 78 derived traits, including the number of antennae of complex-type glycans and levels of fucosylation, bisection, galactosylation, and sialylation. Associations of plasma N-glycomes with age, sex, CD, UC, and IBD-related parameters such as disease location, surgery and medication, level of C-reactive protein, and sedimentation rate were tested by linear and logistic regression. RESULTS: Plasma samples from patients with IBD had a higher abundance of large-size glycans compared with controls, a decreased relative abundance of hybrid and high-mannose structures, lower fucosylation, lower galactosylation, and higher sialylation (α2,3- and α2,6-linked). We could discriminate plasma from patients with CD from that of patients with UC based on higher bisection, lower galactosylation, and higher sialylation (α2,3-linked). Glycosylation patterns were associated with disease location and progression, the need for a more potent medication, and surgery. These results were replicated in a large independent cohort. CONCLUSIONS: We performed high-throughput analysis to compare total plasma N-glycomes of individuals with vs without IBD and to identify patterns associated with disease features and the need for treatment. These profiles might be used in diagnosis and for predicting patients' responses to treatment.

An In-Depth Glycosylation Assay for Urinary Prostate-Specific Antigen.

The concentration of prostate-specific antigen (PSA) in serum is used as an early detection method of prostate cancer (PCa); however, it shows low sensitivity, specificity, and a poor predictive value. Initial studies suggested the glycosylation of PSA to be a promising marker for a more specific yet noninvasive PCa diagnosis. Recent studies on the molecular features of PSA glycosylation (such as antenna modification and core fucosylation) were not successful in demonstrating its potential for an improved PCa diagnosis, probably due to the lack of analytical sensitivity and specificity of the applied assays. In this study, we established for the first time a high-performance PSA Glycomics Assay (PGA), allowing differentiation of α2,6- and α2,3-sialylated isomers, the latter one being suggested to be a hallmark of aggressive types of cancer. After affinity purification from urine and tryptic digestion, PSA samples were analyzed by CE-ESI-MS (capillary electrophoresis-electrospray ionization coupled to mass spectrometry). Based on positive controls, an average interday relative standard deviation of 14% for 41 N-glycopeptides was found. The assay was further verified by analyzing PSA captured from patients' urine samples. A total of 67 N-glycopeptides were identified from the PSA pooled from the patients. In summary, the first PGA successfully established in this study allows an in-depth relative quantitation of PSA glycoforms from urine. The PGA is a promising tool for the determination of potential glycomic biomarkers for the differentiation between aggressive PCa, indolent PCa, and benign prostate hyperplasia in larger cohort studies.

Hinge-Region O-Glycosylation of Human Immunoglobulin G3 (IgG3).

Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out. For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes.

Dopant Enriched Nitrogen Gas Combined with Sheathless Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry for Improved Sensitivity and Repeatability in Glycopeptide Analysis.

Over the last years, numerous strategies have been proposed to enhance both ionization efficiency and spray stability in electrospray ionization (ESI), in particular for nanospray applications. In nano-liquid chromatography-mass spectrometry (nano-LC-ESI-MS), a better ESI performance has been observed when a coaxial gas flow is added around the ESI emitter. Moreover, enrichment of the gas with an organic dopant has led to an improved desolvation and ionization efficiency with an overall enhanced sensitivity. In this study, the use of a dopant enriched nitrogen (DEN)-gas combined with sheathless capillary electrophoresis (CE)-ESI-MS was evaluated for glycopeptide analysis. Using acetonitrile as a dopant, an increased sensitivity was observed compared to conventional sheathless CE-ESI-MS. Up to 25-fold higher sensitivities for model glycopeptides were obtained, allowing for limits of detection unachieved by state-of-the-art nano-LC-ESI-MS. The effect of DEN-gas on the repeatability and intermediate precision was also investigated. When compared to previously reported nano-LC-ESI-MS measurements, similar values were found for CE-ESI-MS with DEN-gas. The enhanced repeatability fosters the use of DEN-gas sheathless CE-ESI-MS in protein glycosylation analysis, where precision is essential. The use of DEN-gas opens new avenues for highly sensitive sheathless CE-ESI-MS approaches in glycoproteomics research, by significantly improving sensitivity and precision.

Human plasma protein N-glycosylation.

Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level.

In-Depth Glycoproteomic Assay of Urinary Prostatic Acid Phosphatase.

Prostate-specific antigen (PSA) is a well-known clinical biomarker in prostate cancer (PCa) diagnosis, but a better test is still needed, as the serum-level-based PSA quantification exhibits limited specificity and comes with poor predictive value. Prior to PSA, prostatic acid phosphatase (PAP) was used, but it was replaced by PSA because PSA improved the early detection of PCa. Upon revisiting PAP and its glycosylation specifically, it appears to be a promising new biomarker candidate. Namely, previous studies have indicated that PAP glycoforms differ between PCa and non-PCa individuals. However, an in-depth characterization of PAP glycosylation is still lacking. In this study, we established an in-depth glycoproteomic assay for urinary PAP by characterizing both the micro- and macroheterogeneity of the PAP glycoprofile. For this purpose, PAP samples were analyzed by capillary electrophoresis coupled to mass spectrometry after affinity purification from urine and proteolytic digestion. The developed urinary PAP assay was applied on a pooled DRE (digital rectal examination) urine sample from nine individuals. Three glycosylation sites were characterized, namely N94, N220, and N333, via N-glycopeptide analysis. Taking sialic acid linkage isomers into account, a total of 63, 27, and 4 N-glycan structures were identified, respectively. The presented PAP glycoproteomic assay will enable the determination of potential glycomic biomarkers for the early detection and prognosis of PCa in cohort studies.