Stretchable Photonic Crystal-Assisted Glycoprotein Identification for Ovarian Cancer Diagnosis.

Photonic crystals with specific wavelengths can realize surface-enhanced excitation and emission intensities of fluorophores and enhance the fluorescence signals of fluorescent molecules. Herein, stretchable photonic crystals with good mechanochromic properties provide continuously adjustable forbidden wavelengths by stretching to change the lattice spacing, with reflectance peaks blue-shifted up to 110 nm to match indicators of different wavelengths and produce differentiated optical enhancement effects. Glycoproteins are significantly identified as clinical markers. However, the wide participation of glycoproteins in various life processes poses enormous complexity and critical challenges for rapid, facile, high-throughput, and accurate clinical analysis or health assessment. In this work, we proposed a stretchable photonic crystal-assisted glycoprotein identification approach for early ovarian cancer diagnosis. Stretchable photonic crystals can provide rich optical information to efficiently identify glycoproteins in complex matrices. A double-indicator fluorescence sensor was designed to respond to the protein trunk and oligosaccharide segment of glycoproteins separately for improved recognition accuracy. Seven typical glycoproteins could be discriminated from proteins, saccharides, or mixture interferents. Clinical ovarian cancer samples for early, intermediate, and advanced ovarian cancer and healthy subjects were verified with 100% accuracy. This strategy of stretchable photonic crystal-assisted glycoprotein identification provides an effective method for accurate, rapid ovarian cancer diagnosis and timely clinical treatment.

Decoding the glycoproteome: a new frontier for biomarker discovery in cancer.

Cancer early detection and treatment response prediction continue to pose significant challenges. Cancer liquid biopsies focusing on detecting circulating tumor cells (CTCs) and DNA (ctDNA) have shown enormous potential due to their non-invasive nature and the implications in precision cancer management. Recently, liquid biopsy has been further expanded to profile glycoproteins, which are the products of post-translational modifications of proteins and play key roles in both normal and pathological processes, including cancers. The advancements in chemical and mass spectrometry-based technologies and artificial intelligence-based platforms have enabled extensive studies of cancer and organ-specific changes in glycans and glycoproteins through glycomics and glycoproteomics. Glycoproteomic analysis has emerged as a promising tool for biomarker discovery and development in early detection of cancers and prediction of treatment efficacy including response to immunotherapies. These biomarkers could play a crucial role in aiding in early intervention and personalized therapy decisions. In this review, we summarize the significant advance in cancer glycoproteomic biomarker studies and the promise and challenges in integration into clinical practice to improve cancer patient care.

High-throughput N-glycoproteomics with fast liquid chromatographic separation.

N-glycosylation is a common protein post translation modification, which has tremendous structure diversity and wide yet delicate regulation of protein structures and functions. Mass spectrometry-based N-glycoproteomics has become a state-of-the-art pipeline for both qualitative and quantitative characterization of N-glycosylation at the intact N-glycopeptide level, providing comprehensive information of peptide backbones, N-glycosites, monosaccharide compositions, sequence and linkage structures. For high-throughput analysis of large-cohort clinic samples, fast and high-performance separation is indispensable. Here we report our development of 1-h liquid chromatography gradient N-glycoproteomics method and accordingly optimized MS parameters. In the benchmark analysis of cancer and paracancerous tissue of hepatocellular carcinoma, 5,218 intact N-glycopeptides were identified, where 422 site- and structure-specific differential N-glycosylation on 145 N-glycoproteins was observed. The method, representing substantial increase of throughput, can be adopted for fast and efficient analysis of N-glycoproteomes at large scale.

High-sensitivity biosensor based on SERS integrated with dendrimer-assisted boronic acid-functionalized magnetic nanoparticles for IL-6 detection in human serum.

High-sensitivity quantitative analysis of sepsis disease markers in circulating blood is essential for sepsis early diagnosis, rapid stratification, and interventional treatment. Herein, a high-sensitivity biosensor combining surface-enhanced Raman spectroscopy (SERS) and functionalized magnetic materials was developed to quantitatively detect interleukin-6 (IL-6), a glycoprotein disease marker closely related to sepsis. First, boronic acid-functionalized magnetic nanomaterials with high adsorption performance were synthesized by utilizing the branched polyethyleneimine to provide many binding sites for boronic acid. Under antibody-free conditions, dendrimer-assisted boronic acid-functionalized magnetic nanomaterials selectively capture glycoproteins in complex biological samples as bio-capture element. Then, a core-shell bimetallic material with plenty of 'hot spots' was designed and synthesized as the enhancement substrate. The 4-Mercaptobenzonitrile (4-MP) with a characteristic peak at 2224 cm-1(Raman-silent region) was embedded as the Raman reporter to form a SERS immune probe with highly efficient electromagnetic enhancement effect, achieving specific recognition and high-sensitivity detection of IL-6 on bio-capture elements. Using this strategy for quantitative analysis of IL-6, a wide detection range (0.5-5000 pg ml-1) and a low detection limit (0.453 pg ml-1) were obtained. Moreover, this method exhibited excellent detection performance for IL-6 in human serum samples, demonstrating its potential promise in screening clinically relevant diseases. The biosensor presented here not only provides a novel and universally applicable sensing strategy for the enrichment and detection of trace glycoprotein disease markers, but also the application of a portable Raman spectrometer provides a more reliable experimental basis for the diagnosis and treatment of major diseases in the clinic or remote and deprived areas.

Leucine-Rich Alpha-2-Glycoprotein (LRG-1) as a Potential Kidney Injury Marker in Kidney Transplant Recipients.

BACKGROUND Kidney transplantation is the treatment of choice for most patients with end-stage renal disease. To improve patient and transplant survival, non-invasive diagnostic methods for different pathologies are important. Leucine-rich alpha-2-glycoprotein (LRG-1) is an innovative biomarker that is elevated in cases of angiogenesis, inflammation, and kidney injury. However, there are limited data about the diagnostic role of LRG-1 in kidney transplant recipients. The aim of this study was to evaluate the association between serum LRG-1, urine LRG-1, and kidney transplant function and injury. MATERIAL AND METHODS We enrolled 35 kidney transplant recipients in the study. LRG-1 in the serum and urine was detected using ELISA. We evaluated the correlation of serum and urine LRG-1 with traditional serum and urine kidney injury markers. RESULTS A higher level of serum LRG-1 correlates with a higher level of urine LRG-1. Serum LRG-1 has a positive correlation with transplant age, serum urea, serum creatinine, serum cystatin C, proteinuria, and fractional excretion of sodium (FENa) and a negative correlation with hemoglobin and estimated glomerular filtration rate (eGFR). Urine LRG-1 has a positive correlation with serum cystatin C, proteinuria, and urine neutrophil gelatinase-associated lipocalin (NGAL). CONCLUSIONS Higher levels of serum and urine LRG-1 are associated with kidney transplant injury and functional deterioration. Thus, LRG-1 might be also as a biomarker for tubular dysfunction in patients after kidney transplantation.

Charting the Proteoform Landscape of Serum Proteins in Individual Donors by High-Resolution Native Mass Spectrometry.

Most proteins in serum are glycosylated, with several annotated as biomarkers and thus diagnostically important and of interest for their role in disease. Most methods for analyzing serum glycoproteins employ either glycan release or glycopeptide centric mass spectrometry-based approaches, which provide excellent tools for analyzing known glycans but neglect previously undefined or unknown glycosylation and/or other co-occurring modifications. High-resolution native mass spectrometry is a relatively new technique for the analysis of intact glycoproteins, providing a "what you see is what you get" mass profile of a protein, allowing the qualitative and quantitative observation of all modifications present. So far, a disadvantage of this approach has been that it centers mostly on just one specific serum glycoprotein at the time. To address this issue, we introduce an ion-exchange chromatography-based fractionation method capable of isolating and analyzing, in parallel, over 20 serum (glyco)proteins, covering a mass range between 30 and 190 kDa, from 150 µL of serum. Although generating data in parallel for all these 20 proteins, we focus the discussion on the very complex proteoform profiles of four selected proteins, i.e., α-1-antitrypsin, ceruloplasmin, hemopexin, and complement protein C3. Our analyses provide an insight into the extensive proteoform landscape of serum proteins in individual donors, caused by the occurrence of various N- and O-glycans, protein cysteinylation, and co-occurring genetic variants. Moreover, native mass intact mass profiling also provided an edge over alternative approaches revealing the presence of apo- and holo-forms of ceruloplasmin and the endogenous proteolytic processing in plasma of among others complement protein C3. We also applied our approach to a small cohort of serum samples from healthy and diseased individuals. In these, we qualitatively and quantitatively monitored the changes in proteoform profiles of ceruloplasmin and revealed a substantial increase in fucosylation and glycan occupancy in patients with late-stage hepatocellular carcinoma and pancreatic cancer as compared to healthy donor samples.

Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery.

Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV's complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein's post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.

Bayesian estimation of sensitivity and specificity of a milk pregnancy-associated glycoprotein ELISA test for pregnancy diagnosis between 23 and 27 days after insemination in Holstein dairy cows.

Pregnancy diagnosis using pregnancy-associated glycoprotein (PAG) ELISA technology in blood or milk samples is validated from 28 d after insemination in dairy cows. The objective of this study was to estimate the sensitivity (Se) and specificity (Sp) of a commercial milk PAG-based ELISA in Holstein dairy cows between 23 and 27 d after insemination. Milk samples (n = 268) from 257 Holstein dairy cows 23 to 27 d after AI were submitted for PAG ELISA testing. Pregnancy status was confirmed by either a second milk PAG ELISA test conducted between 28 and 50 d after insemination (n = 200) or transrectal ultrasonography performed between 28 and 59 d after insemination (n = 68). A Bayesian latent class model was used to compare the paired results from the test at 23 to 27 d after AI test to the reference test. The latent class model typically used for comparing 2 or more imperfect tests was extended to include the possibility of pregnancy loss between the 23 to 27 d test and the reference test. Informative priors for the probability of pregnancy loss, and for the Se and Sp of the PAG and ultrasonography reference tests were obtained from the scientific literature. Estimated median Se and Sp of the PAG ELISA test conducted between 23 and 27 d after AI were 0.98 (95% credible interval 0.93 to 1.0) and 0.98 (0.89 to 1.0), respectively, when using a standardized corrected optical density threshold of 0.15. Although the accuracy of the test under investigation was excellent, more data will be needed to confirm the optimal diagnostic cut point for PAG in milk for early pregnancy diagnosis in this time window. The optimal timing of pregnancy diagnosis will depend on herd-specific logistics and the action to be taken to re-inseminate nonpregnant cows.

Detecting in-solution conformational changes in viral fusogens using tryptophan-induced fluorescence quenching.

Dynamic monitoring of protein conformational changes is necessary to fully understand many biological processes. For example, viral entry and membrane fusion require rearrangement of its viral glycoprotein. We present a step-by-step protocol for site-specific bimane labeling of the influenza-C fusogen to map proximity and conformational movements using tryptophan-induced fluorescence quenching. This protocol is adaptable for other proteins and for protein-protein interaction detection. For complete details on the use and execution of this protocol, please refer to Serrão et al., 2021.

Stanniocalcin 1 is overexpressed in multipotent mesenchymal stromal cells from acute myeloid leukemia patients.

Objectives: Multipotent mesenchymal stromal cells (MSC) play a pivotal role in the bone marrow (BM) niche. Stanniocalcin 1 (STC1) secreted by MSC has been demonstrated to promote the survival of neoplastic cells and was suggested a marker for minimal residual disease of acute myeloid leukemia (AML). Therefore, we evaluated the expression of STC1 in MSC from AML patients (MSCAML) compared to MSC from healthy donors (MSCHD).Methods: Liquid culture assays of MSCAML and MSCHD were performed to compare expansion capacity. Gene expression profiles of MSCAML vs. MSCHD were established. Secretion of STC1 was tested by ELISA in MSCAML vs. MSCHD and expression of STC1 in AML- vs. HD-BM by immunohistochemistry. In addition, co-cultures of AML cells on MSC were initiated and ultrastructural intercellular communication patterns were investigated. Finally, the effect of blocking STC1 on AML cells was evaluated.Results: MSCAML showed significant decreased expansion capacity compared to MSCHD. Gene analysis revealed marked overexpression of STC1 in MSCAML. ELISA and immunohistochemical findings confirmed this observation. Electron microscopy analysis showed reciprocal stimulation between AML cells and MSC. Blockade of STC1 did not significantly affect AML cell proliferation and apoptosis.Discussion: Characteristics of MSC differ depending on whether they originate from AML patients or from HD. STC1 was mostly overexpressed in MSCAML compared to MSCHD. In vitro blockade of STC1, however, was not associated with AML cell proliferation and apoptosis.Conclusion: Differences in expression levels of glycoproteins from MSCAML compared to MSCHD not necessarily assume that these molecules are niche-relevant in leukemic disease.

The Need for Community Standards to Enable Accurate Comparison of Glycoproteomics Algorithm Performance.

Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycoproteomics regarding identification strategy and false discovery rate (FDR) calculation that impedes our examinations. As a case study in the overlap between software, here as a case study, we examine recently published SARS-CoV-2 glycoprotein datasets with four glycoproteomics identification software with their recommended protocols: GlycReSoft, Byonic, pGlyco2, and MSFragger-Glyco. These software use different Target-Decoy Analysis (TDA) forms to estimate FDR and have different database-oriented search methods with varying degrees of quantification capabilities. Instead of an ideal overlap between software, we observed different sets of identifications with the intersection. When clustering by glycopeptide identifications, we see higher degrees of relatedness within software than within glycosites. Taking the consensus between results yields a conservative and non-informative conclusion as we lose identifications in the desire for caution; these non-consensus identifications are often lower abundance and, therefore, more susceptible to nuanced changes. We conclude that present glycoproteomics softwares are not directly comparable, and that methods are needed to assess their overall results and FDR estimation performance. Once such tools are developed, it will be possible to improve FDR methods and quantify complex glycoproteomes with acceptable confidence, rather than potentially misleading broad strokes.

Potential Benefits of Bovine Colostrum in Pediatric Nutrition and Health.

Bovine colostrum (BC), the first milk produced from cows after parturition, is increasingly used as a nutritional supplement to promote gut function and health in other species, including humans. The high levels of whey and casein proteins, immunoglobulins (Igs), and other milk bioactives in BC are adapted to meet the needs of newborn calves. However, BC supplementation may improve health outcomes across other species, especially when immune and gut functions are immature in early life. We provide a review of BC composition and its effects in infants and children in health and selected diseases (diarrhea, infection, growth-failure, preterm birth, necrotizing enterocolitis (NEC), short-bowel syndrome, and mucositis). Human trials and animal studies (mainly in piglets) are reviewed to assess the scientific evidence of whether BC is a safe and effective antimicrobial and immunomodulatory nutritional supplement that reduces clinical complications related to preterm birth, infections, and gut disorders. Studies in infants and animals suggest that BC should be supplemented at an optimal age, time, and level to be both safe and effective. Exclusive BC feeding is not recommended for infants because of nutritional imbalances relative to human milk. On the other hand, adverse effects, including allergies and intolerance, appear unlikely when BC is provided as a supplement within normal nutrition guidelines for infants and children. Larger clinical trials in infant populations are needed to provide more evidence of health benefits when patients are supplemented with BC in addition to human milk or formula. Igs and other bioactive factors in BC may work in synergy, making it critical to preserve bioactivity with gentle processing and pasteurization methods. BC has the potential to become a safe and effective nutritional supplement for several pediatric subpopulations.

A new score including CD43 and CD180: Increased diagnostic value for atypical chronic lymphocytic leukemia.

Moreau score has been used to differentiate chronic lymphocytic leukemia (CLL) from other mature B-cell neoplasms. However, it showed limitations in Asian patients. Therefore, we conducted a new score system replacing CD5 and CD23 with CD43 and CD180 to evaluate its diagnostic value of CLL. 237 untreated samples diagnosed with mature B-cell neoplasms were collected and were randomly divided into an exploratory and a validation cohort by a 2:1 ratio. The expression of CD5, CD19, CD20, CD23, CD43, CD79b, CD180, CD200, FMC7, and surface immunoglobulin (SmIg) were analyzed among all the samples. A proposed score was developed based on the logistic regression model. The sensitivity and specificity of the proposed score were calculated by ROC curves. CD43/CD180, CD200, FMC7, and CD79b were included in our new CLL score, which showed a sensitivity of 91.8% and a specificity of 83.1%. These results were confirmed in a validation cohort with a sensitivity of 90.5% (p = 0.808) and a specificity of 79.5% (p = 0.639). In CD5 negative or CD23 negative CLL group, the new CLL score displayed improved sensitivity of 79.4% compared to Moreau score and CLLflow score (41.2% and 47.1%, respectively). In atypical CLL group, the new CLL score showed improved sensitivity of 84.2% compared to Moreau score and CLLflow score (61.4% and 64.9%, respectively). This proposed atypical CLL score helped to offer an accurate differentiation of CLL from non-CLL together with morphological and molecular methods, particularly in Chinese patients with atypical immunophenotype.

Specific enrichment and glycosylation discrepancy profiling of cellular exosomes using a dual-affinity probe.

A hydrophilic probe is employed to enrich exosomes from three kinds of cancer cells by TiO2-phosphate interaction and exosomal glycoproteins by hydrophilic interaction in succession. The probe performs efficiently in both the enrichment processes. And the analytical results confirm that unique exosomal glycoproteins can distinguish parent exosomes from others.

Application of lectin immobilized on polyHIPE monoliths for bioprocess monitoring of glycosylated proteins.

In-process monitoring of glycosylated protein concentration becomes very important with the introduction of perfusion bioprocesses. Affinity chromatography based on lectins allows selective monitoring when carbohydrates are accessible on the protein surface. In this work, we immobilized lectin on polyHIPE type of monoliths and implemented it for bioprocess monitoring. A spacer was introduced to lectin, which increased binding kinetics toward Fc-fusion protein, demonstrated by bio-layer interferometry. Furthermore, complete desorption using 0.25 M galactose was shown. Affinity column exhibited linearity in the range between 0.5 and 8 mg/ml and flow-unaffected binding for the flow-rates between 0.5 and 8 ml/min. Long-term stability over at least four months period was demonstrated. No unspecific binding of culture media components, including host cell proteins and DNA, was detected. Results obtained by affinity column matched concentration values obtained by a reference method.

Sexually dimorphic perineuronal nets in the rodent and primate reproductive circuit.

Perineuronal nets are extracellular glycoprotein structures that have been found on some neurons in the central nervous system and that have been shown to regulate their structural plasticity. Until now work on perineuronal nets has been focused on their role in cortical structures where they are selectively expressed on parvalbumin-positive neurons and are reported to restrict the experience-dependent plasticity of inhibitory afferents. Here, we examined the expression of perineuronal nets subcortically, showing that they are expressed in several discrete structures, including nuclei that comprise the brain network controlling reproductive behaviors (e.g., mounting, lordosis, aggression, and social defense). In particular, perineuronal nets were found in the posterior dorsal division of the medial amygdala, the medial preoptic nucleus, the posterior medial bed nucleus of the stria terminalis, the ventrolateral ventromedial hypothalamus and adjacent tuberal nucleus, and the ventral premammillary nucleus in both the mouse and primate brain. Comparison of perineuronal nets in male and female mice revealed a significant sexually dimorphic expression, with expression found prominently on estrogen receptor expressing neurons in the medial amygdala. These findings suggest that perineuronal nets may be involved in regulating neural plasticity in the mammalian reproductive system.

Leucine-rich alpha-2 glycoprotein is a potential biomarker to monitor disease activity in inflammatory bowel disease receiving adalimumab: PLANET study.

BACKGROUND: This multicenter prospective study (UMIN000019958) aimed to evaluate the usefulness of serum leucin-rich alpha-2 glycoprotein (LRG) levels in monitoring disease activity in inflammatory bowel disease (IBD). METHODS: Patients with moderate-to-severe IBD initiated on adalimumab therapy were enrolled herein. Serum LRG, C-reactive protein (CRP), and fecal calprotectin (fCal) levels were measured at week 0, 12, 24, and 52. Colonoscopy was performed at week 0, 12, and 52 for ulcerative colitis (UC), and at week 0, 24, and 52 for Crohn's disease (CD). Endoscopic activity was assessed using the Simple Endoscopic Score for Crohn's Disease (SES-CD) for CD and the Mayo endoscopic subscore (MES) for UC. RESULTS: A total of 81 patients was enrolled. Serum LRG levels decreased along with improvements in clinical and endoscopic outcomes upon adalimumab treatment (27.4 ± 12.6 µg/ml at week 0, 15.5 ± 7.7 µg/ml at week 12, 15.7 ± 9.6 µg/ml at week 24, and 14.5 ± 6.8 µg/ml at week 52), being correlated with endoscopic activity at each time point (SES-CD: r = 0.391 at week 0, r = 0.563 at week 24, r = 0.697 at week 52; MES: r = 0.534 at week 0, r = 0.429 at week 12, r = 0.335 at week 52). Endoscopic activity better correlated with LRG compared to CRP and fCal on pooled analysis at all time points (SES-CD: LRG: r = 0.636, CRP: r = 0.402, fCal: r = 0.435; MES: LRG: r = 0.568, CRP: 0.389, fCal: r = 0.426). CONCLUSIONS: Serum LRG is a useful biomarker of endoscopic activity both in CD and UC during the adalimumab treatment.

Benchmark of site- and structure-specific quantitative tissue N-glycoproteomics for discovery of potential N-glycoprotein markers: a case study of pancreatic cancer.

Pancreatic cancer is a highly malignant tumor of the digestive tract that is difficult to diagnose and treat. It is more common in developed countries and has become one of the main causes of death in some countries and regions. Currently, pancreatic cancer generally has a poor prognosis, partly due to the lack of symptoms in the early stages of pancreatic cancer. Therefore, most cases are diagnosed at advanced stage. With the continuous in-depth research of glycoproteomics in precision medical diagnosis, there have been some reports on quantitative analysis of cancer-related cells, plasma or tissues to find specific biomarkers for targeted therapy. This research is based on the developed complete N-linked glycopeptide database search engine GPSeeker, combined with liquid-mass spectrometry and stable diethyl isotope labeling, providing a benchmark of site- and structure-specific quantitative tissue N-glycoproteomics for discovery of potential N-glycoprotein markers. With spectrum-level FDR ≤1%, 20,038 intact N-Glycopeptides corresponding to 4518 peptide backbones, 228 N-glycan monosaccharide compositions 1026 N-glycan putative structures, 4460 N-glycosites and 3437 intact N-glycoproteins were identified. With the criteria of ≥1.5-fold change and p value<0.05, 52 differentially expressed intact N-glycopeptides (DEGPs) were found in pancreatic cancer tussues relative to control, where 38 up-regulated and 14 down-regulated, respectively.

Determination of Isomeric Glycan Structures by Permethylation and Liquid Chromatography-Mass Spectrometry (LC-MS).

The existence of glycans in isomeric forms is responsible for the multifariousness of their properties and biological functions. Their altered expression has been associated with various diseases and cancers. Analysis of native glycans is not very sensitive due to the low ionization efficiency of glycans. These facts necessitate their comprehensive structural studies and establishes a high demand for sensitive and reliable techniques. In this chapter, we discuss the strategies for effective separation and identification of permethylated isomeric glycans. The sample preparation for permethylated glycans derived from model glycoproteins and complex biological samples, analyzed using LC-MS/MS, is delineated. We introduce protein extraction and release of glycans, followed by strategies to purify the released glycans, which are reduced and permethylated to improve ionization efficiency and stabilize sialic acid residues. High-temperature LC-based separation on PGC (porous graphitized carbon) column is conducive to isomeric separation of glycans and allows their sensitive identification and quantification using MS/MS.