Human Serum Glycoproteins Research Articles

Site-Specific Analysis of Core and Antenna Fucosylation on Serum Glycoproteins.

Fucosylation is an important structural feature of glycans and plays an essential role in the regulation of glycoprotein functions. Fucosylation can be classified into core- (CF) and antenna-fucosylation (AF, also known as (sialyl-) Lewis) based on the location on N-glycans, and they perform distinct biological functions. In this study, core- and antenna-fucosylated N-glycans on human serum glycoproteins that hold great clinical application values were systematically characterized at the site-specific level using StrucGP combined with the recently developed fucosylation assignment method. The results showed that fucosylation was widely distributed on serum glycoproteins, with 50% of fucosylated glycopeptides modified by AF N-glycans, 37% by CF N-glycans, and 13% by dual-fucosylated N-glycans. Interestingly, CF and AF N-glycans preferred to modify different groups of serum glycoproteins with different tissue origins and were involved in distinctive biological processes. Specifically, AF N-glycoproteins are mainly from the liver and participated in complement activation, blood coagulation, and endopeptidase activities, while CF N-glycoproteins originate from diverse tissues and are mainly involved in cell adhesion and signaling transduction. These data further enhanced our understanding of fucosylation on circulation glycoproteins.

Janus nanozyme based satellite structure immunosandwich colorimetric strategy for glycoproteins visual detection

Glycoproteins are reliable biomarkers for early cancer detection, and the timely response to these biomarkers could improve the survival rate and reduce the medical care burden. However, conventional detection technologies such as immune-based assays often need natural antibody, complex sample pretreatments and long analysis time, which failing to provide the rapid turnaround times. To address this problem, a janus gold nanoparticles nanozyme (J-GNPsNE) linked magnetic titanium dioxide molecularly imprinted particles (MTi-MIP) satellite structure immunosandwich colorimetric strategy (SS-ICS) is proposed to rapid detect glycoproteins in human serum for the first time. The unique detection mode of the strategy relies on the sandwich-like assembly of molecularly imprinted nanoparticles, glycoprotein and nanozyme. This enabled to establish a point-of-care, labor-saving, easy-to-use and noninvasive serum glycoprotein biomarkers detection tool for efficient early diagnosis of cancer. By taking transferrin (TrF) as an example, the proposed strategy can exhibits great performance with a low detection limit (0.052 μg/mL) and a short analysis time (40 min), which is much better than most of the previous methods. Our presented strategy can be easily generalized for specific detection other types of glycoproteins by changing the template, thereby facilitating the serum screening for early cancer diagnosis in non-specialized laboratory infrastructure.

Quantitative detection of α1-acid glycoprotein (AGP) level in blood plasma using SERS and CNN transfer learning approach

Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive tool in medical diagnostics and bioanalysis fields, aimed at the qualitative detection of relevant biomolecules. However, quantitative SERS analysis of complex (bio)samples is a more challenging and, in many cases, almost impossible task, requiring functional SERS substrates or advanced spectral data analysis. In this work, we propose the combination of a functional SERS substrate, capable of trapping target biomolecules, with CNN transfer learning for quantitative detection of the relevant α1-acid glycoprotein (AGP, also known as orosomucoid) in human serum. As a SERS substrate, the plasmonic gold grating was functionalized with boronic acid moieties to entrap target AGP. The functionality of the substrate was tested on two model solutions: a solution containing saccharides as competing molecules and human serum with added AGP, which is close to real samples. The convolution neural network (CNN) was previously trained on a huge number of (bio)samples. Then CNN transfer learning was used to quantify AGP concentration in model samples, as well as in human serum. Developed strategy is able to identify the alarming increase of AGP concentration in an express and medically decentralized way, on short time and under lack of spectral data. Generally, the proposed combination of SERS and machine transfer learning could be expanded to a range of alternative cases, where the collection of real samples is restricted and can be substituted by the measurements of similar model systems, without loss of analysis reliability.

Changes of IgG N-Glycosylation in Thyroid Autoimmunity: The Modulatory Effect of Methimazole in Graves' Disease and the Association With the Severity of Inflammation in Hashimoto's Thyroiditis.

The N-glycome of immunoglobulin G (IgG), the most abundant glycoprotein in human blood serum, reflects pathological conditions of autoimmunity and is sensitive to medicines applied in disease therapy. Due to the high sensitivity of N-glycosylation, the IgG N-glycan profile may serve as an indicator of an ongoing inflammatory process. The IgG structure and its effector functions are strongly dependent on the composition of N-glycans attached to the Fc fragment, and the binding of antigens is regulated by Fab sugar moieties. Because of the crucial role of N-glycans in IgG function, remodeling of its N-oligosaccharides can induce pathological changes that ultimately contribute to the development of autoimmunity; restoration of their physiological structure is critical to the reduction of disease symptoms. Our recently published data have shown that the pathology of autoimmune thyroid diseases (AITDs), including Hashimoto’s thyroiditis (HT) and Graves’ disease (GD), is accompanied by alterations of the composition of IgG N-glycans. The present study is a more in-depth investigation of IgG glycosylation in both AITDs, designed to determine the relationship between the severity of thyroid inflammation and IgG N-glycan structures in HT, and to assess the impact of immunosuppressive therapy on the N-glycan profile in GD patients. The study material consisted of human serum samples collected from donors with elevated anti-thyroglobulin (Tg) and/or anti-thyroperoxidase (TPO) IgGs without symptoms of hypothyroidism (n=68), HT patients characterized by high autoantibody titers and advanced destruction of the thyroid gland (n=113), GD patients with up-regulated IgG against thyroid-stimulating hormone receptor (TSHR) before (n=62) and after (n=47) stabilization of TSH level as a result of methimazole therapy (study groups), and healthy donors (control group, n=90). IgG was isolated from blood serum using protein G affinity chromatography. N-glycans were released from IgG by PNGase F digestion and analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) after 2-aminobenzamide (2-AB) labeling. UPLC-MS chromatograms were integrated into 25 peaks (GP) in the Waters UNIFI Scientific Information System, and N-glycans were assigned based on the glucose unit values and mass-to-charge ratios (m/z) of the detected ions. The Kruskal-Wallis non-parametric test was used to determine the statistical significance of the results (p<0.05). The obtained results suggest that modifications of IgG sialylation, galactosylation and core-fucosylation are associated with the severity of HT symptoms. Methimazole therapy implemented in GD patients affected the IgG N-glycan profile; as a result, the content of the sialylated and galactosylated oligosaccharides with core fucose differed after treatment. Our results suggest that N-glycosylation of IgG undergoes dynamic changes during the intensification of thyroiditis in HT, and that in GD autoimmunity it is affected significantly by immunosuppressive therapy.

Coagulation factor IX analysis in bioreactor cell culture supernatant predicts quality of the purified product

Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Bioreactor operating conditions can impact rFIX production and post-translational modifications (PTMs). With the goal of optimizing rFIX production, we developed a suite of Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics methods and used these to investigate rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. We detail the dynamics of site-specific PTM occupancy and structure on rFIX during production, which correlated with the efficiency of purification and the quality of the purified product. We identified new PTMs in rFIX near the GLA domain which could impact rFIX GLA-dependent purification and function. Our workflows are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.

IgG N-glycans.

Glycosylation, one of the most common post-translational modifications in mammalian cells, impacts many biological processes such as cell adhesion, proliferation and differentiation. As the most abundant glycoprotein in human serum, immunoglobulin G (IgG) plays a vital role in immune response and protection. There is a growing body of evidence suggests that IgG structure and function are modulated by attached glycans, especially N-glycans, and aberrant glycosylation is associated with disease states. In this chapter, we review IgG glycan repertoire and function, strategies for profiling IgG N-glycome and recent studies. Mass spectrometry (MS) based techniques are the most powerful tools for profiling IgG glycome. IgG glycans can be divided into high-mannose, biantennary complex and hybrid types, modified with mannosylation, core-fucosylation, galactosylation, bisecting GlcNAcylation, or sialylation. Glycosylation of IgG affects antibody half-life and their affinity and avidity for antigens, regulates crystallizable fragment (Fc) structure and Fcγ receptor signaling, as well as antibody effector function. Because of their critical roles, IgG N-glycans appear to be promising biomarkers for various disease states. Specific IgG glycosylation can convert a pro-inflammatory response to an anti-inflammatory activity. Accordingly, IgG glycoengineering provides a powerful approach to potentially develop effective drugs and treat disease. Based on the understanding of the functional role of IgG glycans, the development of vaccines with enhanced capacity and long-term protection are possible in the near future.

Human anti-α-fucose antibodies are xenoreactive toward GGTA1/CMAH knockout pigs.

Progress has been made in overcoming antibody-mediated rejection of porcine xenografts by deleting pig genes that produce unique carbohydrate epitopes. Pigs deficient in galactose α-1,3 galactose (gene modified: GGTA1) and neu5Gc (gene modified: CMAH) have reduced levels of human antibody binding. Previously we identified α-fucose as a glycan that was expressed in high levels on cells of GGTA1/CMAH KO pigs. To validate the α-fucose phenotype observed previously we compared lectin affinity toward human and pig serum glycoproteins by dot blot analysis and confocal microscopy. Human anti-fucose antibody isolated by affinity chromatography was tested for specificity to L-fucose by custom macroarray. The affinity and cytotoxicity of the isolated human anti-fucose antibody toward human and GGTA1/CMAH KO pig PBMCs was determined by flow cytometry. Dot blot and confocal analysis support out previous findings that α-fucose is more highly expressed in pigs than humans. Pig kidney glomeruli and tubules contain abundant α-fucose and may represent focal sites for anti-α-fucose antibody binding. The Isolated human anti-fucose IgA, IgG and IgM bound to GGTA1/CMAH KO pig PBMC and were cytotoxic. Interestingly, the isolated human IgG cross reacted with the methyl pentose, L-rhamnose. Human anti-fucose antibody bound and was cytotoxic to GGTA1/CMAH KO pig peripheral blood monocytes. We have shown that α-fucose is an abundant target for cytotoxic human antibody in the organs of genetically modified pigs important to xenotransplantation.

Identifying Sialylation Linkages at the Glycopeptide Level by Glycosyltransferase Labeling Assisted Mass Spectrometry (GLAMS).

Precise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics and an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of α2,3/α2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, we developed an innovative glycosyltransferase labeling assisted mass spectrometry (GLAMS) strategy. After specific enzymatic labeling, oxonium ions from higher-energy C-trap dissociation (HCD) fragmentation of α2,3-sailoglycopeptides then generate unique reporters to distinctly differentiate those of α2,6-sailoglycopeptide isomers. With this strategy, a total of 1236 linkage-specific sialoglycopeptides were successfully identified from 161 glycoproteins in human serum.

Large-scale Identification of N-linked Intact Glycopeptides in Human Serum using HILIC Enrichment and Spectral Library Search

Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of N-linked intact glycopeptides from N-linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control. Serum proteins were firstly separated into low-abundance and high-abundance proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and a portion of the enriched N-linked intact glycopeptides were processed by Peptide-N-Glycosidase F (PNGase F) to generate N-linked deglycopeptides. Both N-linked intact glycopeptides and deglycopeptides were analyzed by LC-MS/MS. From N-linked deglycopeptides data sets, 764 N-linked glycoproteins, 1699 N-linked glycosites and 3328 unique N-linked deglycopeptides were identified. Four types of N-linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the N-linked deglycopeptides. The spectra of these N-linked deglycopeptides were utilized for N-linked deglycopeptides library construction and identification of N-linked intact glycopeptides. A database containing 739 N-glycan masses was constructed and utilized during spectral library search for the identification of N-linked intact glycopeptides. In total, 526 N-linked glycoproteins, 1036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level.

Purification of N- and O-glycans and their derivatives from biological samples by the absorbent cotton hydrophilic chromatographic column

Mass spectrum (MS) is one of the most commonly used tools for qualitative and quantitative analysis of glycans. However, due to the complexity of biological samples and the low ionization efficiency of glycans, these need to be purified and derivatized prior to MS analysis. Existing purification strategies require a combination of multiple methods and are cumbersome to operate. Here, we propose a new method for the purification of glycoprotein N/O-glycans and their derivatives using a hand-packed absorbent cotton hydrophilic interaction chromatography column (HILIC). The method's reliability and applicability were verified by purifying N/O-glycans and the derivatives of standard glycoproteins, such as chicken albumin and porcine stomach mucin. Stable isotope labelling was used to compare the glycans’ recovery following different purification methods. Absorbent cotton HILIC was also successfully applied for the analysis of human serum and fetal bovine serum glycoprotein N-glycans. Finally, testing revealed high binding capacity (9 mg/g−1 maltohexaose/absorbent cotton) and good recovery (average recovery was 91.7%) of glycans. Compared with traditional procedures, the proposed purification method offers considerable advantages, such as simplicity, high efficiency, economy, universality, and broad applicability for the pretreatment of glycans and their derivatives in biological samples prior to MS analysis.

A Novel Mass Spectrometry Platform for Multiplexed N-Glycoprotein Biomarker Discovery from Patient Biofluids by Antibody Panel Based N-Glycan Imaging.

A new platform for N-glycoprotein analysis from serum that combines matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) workflows with antibody slide arrays is described. Antibody panel based (APB) N-glycan imaging allows for the specific capture of N-glycoproteins by antibodies on glass slides and N-glycan analysis in a protein-specific and multiplexed manner. Development of this technique has focused on characterizing two abundant and well-studied human serum glycoproteins, alpha-1-antitrypsin and immunoglobulin G. Using purified standard solutions and 1 μL samples of human serum, both glycoproteins can be immunocaptured and followed by enzymatic release of N-glycans. N-Glycans are detected with a MALDI FT-ICR mass spectrometer in a concentration-dependent manner while maintaining specificity of capture. Importantly, the N-glycans detected via slide-based antibody capture were identical to that of direct analysis of the spotted standards. As a proof of concept, this workflow was applied to patient serum samples from individuals with liver cirrhosis to accurately detect a characteristic increase in an IgG N-glycan. This novel approach to protein-specific N-glycan analysis from an antibody panel can be further expanded to include any glycoprotein for which a validated antibody exists. Additionally, this platform can be adapted for analysis of any biofluid or biological sample that can be analyzed by antibody arrays.

An integrated approach for comprehensive profiling and quantitation of IgG-Fc glycopeptides with application to rheumatoid arthritis

Glycosylation plays an important role in the maintenance of the structure and function of glycoproteins, while aberrant protein glycosylation is correlated with various diseases. Human immunoglobulin G (IgG), which is composed of four subclasses (IgG1, IgG2, IgG3 and IgG4), is one of the most dominant and significant glycoprotein in human serum. The glycosylation on IgG-Fc moiety is known to be alternated with various physiological and pathological states. We herein report an integrated approach for comprehensive profiling and quantitation of IgG-Fc glycopeptides. Firstly, IgG N-glycans were profiled by using mAb-Glyco chip quadrupole time-of-flight mass spectrometry (Q-TOF-MS), resulting characterization of 87 N‑glycans originating from 29 different oligosaccharide compositions. Secondly, subclass-specific glycopeptides were identified on the basis of high-resolution MS and MS/MS data by using ultra high performance liquid chromatography (UHPLC) coupled to Q-TOF-MS. As a result, 83 IgG-Fc glycopeptides from human serum, including 17 sialylated glycopeptides, were identified. In addition, a quantitation method with high sensitivity and repeatability was established by using UHPLC triple quadrupole (QQQ) MS. We applied this approach to carry out quantitative analysis of IgG glycosylation in RA patients. Finally, 36 potential glycopeptide biomarkers, including 13 species from IgG1, 12 species from IgG2/3 and 11 species from IgG4 were identified.

Multipronged ESI-MS Approach for Studying Glycan-Binding Protein Interactions with Glycoproteins.

A multipronged electrospray ionization mass spectrometry (ESI-MS) approach for investigating glycan-mediated interactions between water-soluble glycan-binding proteins (GBPs) and glycoproteins (GPs) is described. First, the catch-and-release (CaR)-ESI-MS assay, carried with ion mobility separation prior to GBP "release" (i.e., CaRIMS-ESI-MS), is employed to rapidly identify GBP-GP binding in solution. The apparent affinity ( Ka) of the GBP for the GP is then measured using the competitive proxy ligand-ESI-MS binding assay. Finally, N-glycans, enzymatically released as free oligosaccharides from the GP, are screened against the GBP using ESI-MS to identify the glycans that are recognized by the GBP. Measurements performed at multiple GBP concentrations allow for the affinities of released N-glycans (grouped as compositional isomers) to be ranked. Implementation of the approach is illustrated using the known interactions between a C-terminal domain fragment of human galectin-3 (hGal-3C) and three human serum GPs, α-1-acid glycoprotein (AGP), haptoglobin phenotype 1-1 (Hp1-1) and α-2-macroglobulin (α2M). Specific binding of hGal-3C to each GP was successfully detected by CaRIMS-ESI-MS; no binding was detected for a noninteracting reference protein, which served as a negative control. The Ka measured by proxy ligand-ESI-MS for AGP, Hp1-1 and α2M (4 × 105 M-1, 2 × 105 M-1 and 3 × 105 M-1, respectively) are consistent with the reported apparent affinities of hGal-3 for serum GPs and their N-glycans. Screening the N-glycan libraries of each of the GPs against hGal-3C identified ligands corresponding to a total of 20 different saccharide compositions with sialylated bi-, tri-, and tetra-antennary structures. The results of binding measurements performed at two different hGal-3C concentrations revealed that all of the N-glycan ligands exhibit affinities ≥104 M-1.

High-throughput detection of low abundance sialylated glycoproteins in human serum by TiO2 enrichment and targeted LC-MS/MS analysis: application to a prostate cancer sample set.

Glycopeptide enrichment can be a strategy to allow the detection of peptides belonging to low abundance proteins in complex matrixes such as blood serum or plasma. Though several glycopeptide enrichment protocols have shown excellent sensitivities in this respect, few reports have demonstrated the applicability of these methods to relatively large sample cohorts. In this work, a fast protocol based on TiO2 enrichment and highly sensitive mass spectrometric analysis by Selected Reaction Monitoring (SRM) has been applied to a cohort of serum samples from prostate cancer and benign prostatic hyperplasia patients in order to detect low abundance proteins in a single LC-MS/MS analysis in nanoscale format, without immunodepletion or peptide fractionation. A peptide library of over 700 formerly N-glycosylated peptides was created by data dependent analysis. Then, 16 medium to low abundance proteins were selected for detection by single injection LC-MS/MS based on selected-reaction monitoring. Results demonstrated the consistent detection of the low-level proteins under investigation. Following label-free quantification, four proteins (Adipocyte plasma membrane-associated protein, Periostin, Cathepsin D and Lysosome-associated membrane glycoprotein 2) were found significantly increased in prostate cancer sera compared to the control group. Graphical abstract ᅟ.

IgG glycosylation in autoimmune diseases

Immunoglobulin G (IgG) is the most abundant glycoprotein in human serum. All IgG subclasses have a single-conserved N-linked glycosylation site at Asn297 of the heavy chain and 10–30% of IgGs are N-glycosylated also in a Fab region. N-glycans of Fc are sialylated and fucosylated biantennary complex-type structures. Glycosylation plays a key role in antibody function, and IgG N-glycans are essential for the proper activity of the immune system. Fc glycans are important for IgG effector functions, whereas Fab oligosaccharides modulate antigen binding. Glycosylation changes of IgG are associated with the development of various human diseases, including autoimmune states. The modification of one sugar moiety in N-glycan structure may result in the stimulation or suppression of immune response. The lack of core fucose leads to the enhancement of pro-inflammatory activity, whereas an increase of sialylation determines immunosuppressive properties of IgG. The contribution of IgG Fc glycosylation changes has been demonstrated in the pathogenesis of rheumatoid arthritis, lupus erythematosus and Crohn’s disease. A decrease in IgG galactosylation and sialylation, found in these diseases, activates effector cells and triggers inflammatory reactions. A detailed analysis of changes in IgG glycosylation and their effects on the development of autoimmune diseases is important in the treatment of these diseases. IgGs with modified α2,6-sialylation are used as therapeutic antibodies with anti-inflammatory properties. Numerous studies on IgG glycosylation have provided evidence of the role of this post-translational modification in the proper functioning of antibodies and the importance of changes in the structure of IgG glycans, mainly incomplete galactosylation and desialylation, in the pathogenesis of many diseases. The continuation of these studies may contribute to explaining the mechanisms of autoimmunity that is still poorly understood.

Disposable carbon nanotube scaffold films for fast and reliable assessment of total α1-acid glycoprotein in human serum using adsorptive transfer stripping square wave voltammetry.

Alpha-1-acid glycoprotein (AGP) is a serum glycoprotein whose levels are increased two or three times during disease or injury. This makes it a potential biomarker for inflammatory bowel diseases and sepsis. Consequently, fast, simple, and cheap analytical methods for prognosis, diagnosis, and follow-up of these diseases are demanded. In this work, we propose a simple electrochemical approach based on carbon nanotubes scaffold films (CNSFs) for total AGP determination in serum samples. Firstly, AGP is labeled with an electrochemical tag (osmium(VI) complex), and then the total amount of AGP is quantified by adsorptive transfer stripping square wave voltammetry (AdTSWV). Multi-walled carbon nanotubes scaffold films (MWSFs) yielded the best analytical performance in terms of sensitivity with a good limit of detection of 0.6mgL-1 for AGP determination in serum samples, in less than 20min. A simplified AGP calibration and its sequential serum sample analysis strategy with good accuracy (81%) and excellent reproducibility (RSD < 1%) was additionally proposed to meet the point-of-care/needs requirements. Graphical abstract Multi-walled carbon nanotubes scaffold films for total AGP determination on disposable platforms integrating single-point calibration and sequential sample analysis.

Specificity and utility of SubB2M, a new N-glycolylneuraminic acid lectin

The B subunit of the subtilase cytotoxin (SubB) recognises N-glycolylneuraminic acid (Neu5Gc) containing glycans, the most prominent form of aberrant glycosylation in human cancers. We have previously engineered SubB by construction of a SubBΔS106/ΔT107 mutant (SubB2M) for greater specificity and enhanced recognition of Neu5Gc containing glycans. In this study, we further explore the utility of SubB2M as a Neu5Gc lectin by showing its improved specificity and recognition for Neu5Gc containing glycans over the wild-type SubB protein and an anti-Neu5Gc IgY antibody in a N-acetylneuraminic acid (Neu5Ac)/Neu5Gc glycan array and by surface plasmon resonance. Far-western blot analysis showed that SubB2M preferentially binds to bovine serum glycoproteins over human serum glycoproteins. SubB2M was also able to detect Neu5Gc containing bovine glycoproteins spiked into normal human serum with greater sensitivity than the wild-type SubB and the anti-Neu5Gc IgY antibody. These results suggest that SubB2M will be a useful tool for the testing of serum and other bodily fluids for cancer diagnosis and prognosis.

An endoglycosidase-assisted LC-MS/MS-based strategy for the analysis of site-specific core-fucosylation of low-concentrated glycoproteins in human serum using prostate-specific antigen (PSA) as example

Recently, site-specific fucosylation of glycoproteins has attracted attention as it can be associated with several types of cancers including prostate cancer. However, individual glycoproteins, which might serve as potential cancer markers, often are very low-concentrated in complex serum matrices and distinct glycan structures are hard to detect by immunoassays. Here, we present a mass spectrometry-based strategy for the simultaneous analysis of core-fucosylated and total prostate-specific antigen (PSA) in human serum in the low ng/ml concentration range. Sample preparation comprised an immunoaffinity capture step to enrich total PSA from human serum using anti-PSA antibody coated magnetic beads followed by consecutive two-step on-bead partial deglycosylation with endoglycosidase F3 and tryptic digestion prior to LC-MS/MS analysis. The method was shown to be linear from 0.5 to 60 ng/ml total PSA concentrations and allows the simultaneous quantification of core-fucosylated PSA down to 1 ng/ml and total PSA lower than 0.5 ng/ml. The imprecision of the method over two days ranged from 9.7–23.2% for core-fucosylated PSA and 10.3–18.3% for total PSA depending on the PSA level. The feasibility of the method in native sera was shown using three human specimens. To our knowledge, this is the first MS-based method for quantification of core-fucosylated PSA in the low ng/ml concentration range in human serum. This method could be used in large patient cohorts as core-fucosylated PSA may be a diagnostic biomarker for the differentiation of prostate cancer and other prostatic diseases, such as benign prostatic hyperplasia (BPH). Furthermore, the described strategy could be used to monitor potential changes in site-specific core-fucosylation of other low-concentrated glycoproteins, which could serve as more specific markers (“marker refinement”) in cancer research.

LC-MS/MS of permethylated N-glycans derived from model and human blood serum glycoproteins.

LC-MS/MS is one of the most powerful tools for N-glycan structure elucidation; however, it is still challenging to identify some glycan structures with low abundance. In this study, we investigated the chromatographic behavior of permethylated N-glycans. The relationship between retention times versus molecular weight of dextran, dextrin, and model glycans was investigated. Also, the nonpolar surface area of glycans was calculated and compared to their experimental retention times. Both retention time and nonpolar surface area trends are similar when the intermolecular interaction is included in the calculation. Moreover, retention time corresponds to glycan types and branch types. The N-glycans analysis model, which combines high mass accuracy and retention time, was applied to confirm serum N-glycans. In total, there were 78 N-glycan compositions identified. A linear relationship between retention times and molecular weights were observed for each subgroup of glycan structures, for example, R(2) value for complex N-glycans was determined to be > 0.98. Moreover, the retention time could be further applied to distinguish between structural isomers as well as linkage isomers. MS/MS data were used to confirm the structural isomers.

Hydrazino-s-triazine based labelling reagents for highly sensitive glycan analysis via liquid chromatography–electrospray mass spectrometry

Labelling strategy plays an important role in mass spectrometry (MS) based glycan analysis due to the high hydrophilicity and low ionization efficiency of glycans. Ten hydrazino-s-triazine based labelling reagents were synthesized under facile and controllable conditions for highly sensitive liquid chromatography–electrospray mass spectrometry glycan analysis in this work. Attached to N-glycans through non-reductive reactions, these new labelling reagents were evaluated in aspect of the differently enhanced glycan response to mass spectrometry. Three of the ten labelling reagents demonstrated to be reliable and remarkable for glycan analysis with satisfactory linearity and lowered limits of detection using maltoheptaose (DP7) as model. Furthermore, the most optimal labelling reagent was taken as an example for highly sensitive profiling of N-linked glycans both cleaved from chicken avidin and glycoproteins in human serum, indicating prospective availability for these labelling reagents in frontier of glycomics researches.