Total Plasma N‑Glycome Changes during Pregnancy

During pregnancy, the mother faces a major immunological challenge. Most of the major plasma proteins have important immunological functions, and altered levels of these major proteins have been reported during pregnancy, potentially providing immunosuppression. A large number of the high abundance plasma proteins are post-translationally modified by N-glycans, and while it is now understood that these glycans may also affect the immunological functions, their pattern has not been studied in relation to pregnancy. Here, the N-glycosylation profile of 32 pregnant women was determined over the course of their pregnancy using a multiplexed CGE-LIF method. Moreover, for 6 women, the glycosylation profiles of the proteins IgG, IgA, and alpha1-antitrypsin were monitored. For total plasma, 16 glycan signals showed differential expression during pregnancy. In general the levels of largely sialylated bi-, tri-, and tetra-antennary glycans were increased during pregnancy, while biantennary glycans with no more than one sialic acid were decreased. Similarly altered glycosylation profiles were observed for the individual proteins IgG, with a decrease of digalactosylated biantennary glycans after delivery, and alpha1-antitrypsin, on which increased levels of triantennary glycans were observed during pregnancy. Overall, these results show altered glycosylation profiles both for total plasma glycoproteins and on individual proteins during pregnancy, which may contribute to immunosuppression and have other biological functions

Label-Free Absolute Quantitation of Oligosaccharides Using Multiple Reaction Monitoring

An absolute quantitation method for measuring free human milk oligosaccharides (HMOs) in milk samples was developed using multiple reaction monitoring (MRM). To obtain the best sensitivity, the instrument conditions were optimized to reduce the source and postsource fragmentation prior to the quadrupole transmission. Fragmentation spectra of HMOs using collision-induced dissociation were studied to obtain the best characteristic fragments. At least two MRM transitions were used to quantify and identify each structure in the same run. The fragment ions corresponded to the production of singly charged mono-, di-, and trisaccharide fragments. The sensitivity and accuracy of the quantitation using MRM were determined, with the detection limit in the femtomole level and the calibration range spanning over 5 orders of magnitude. Seven commercial HMO standards were used to create calibration curves and were used to determine a universal response for all HMOs. The universal response factor was used to estimate absolute amounts of other structures and the total oligosaccharide content in milk. The quantitation method was applied to 20 human milk samples to determine the variations in HMO concentrations from women classified as secretors and nonsecretors, a phenotype that can be identified by the concentration of 2′-fucosylation in their milk

Glycoproteomic Analysis of Malignant Ovarian Cancer Ascites Fluid Identifies Unusual Glycopeptides

Ovarian cancer is a major cause of cancer mortality among women, largely due to late diagnosis of advanced metastatic disease. More extensive molecular analysis of metastatic ovarian cancer is needed to identify post-translational modifications of proteins, especially glycosylation that is particularly associated with metastatic disease to better understand the metastatic process and identify potential therapeutic targets. Glycoproteins in ascites fluid were enriched by affinity binding to lectins (ConA or WGA) and other affinity matrices. Separate glycomic, proteomic, and glycopeptide analyses were performed. Relative abundances of different N-glycan groups and proteins were identified from ascites fluids and a serum control. Levels of biomarkers CA125, MUC1, and fibronectin were also monitored in OC ascites samples by Western blot analysis. N-Glycan analysis of ascites fluids showed the presence of large, highly fucosylated and sialylated complex and hybrid glycans, some of which were not observed in normal serum. OC ascites glycoproteins, haptoglobin, fibronectin, lumican, fibulin, hemopexin, ceruloplasmin, alpha-1-antitrypsin, and alpha-1-antichymotrypsin were more abundant in OC ascites or not present in serum control samples. Further glycopeptide analysis of OC ascites identified N- and O-glycans in clusterin, hemopexin, and fibulin glycopeptides, some of which are unusual and may be important in OC metastasis

Protein-Specific Differential Glycosylation of Immunoglobulins in Serum of Ovarian Cancer Patients

Previous studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian cancer; however, it was unclear to which proteins these glycans belong. We hypothesize that protein-specific glycosylation profiles of the glycans may be more informative of ovarian cancer and can provide insight into biological mechanisms underlying glycan aberration in serum of diseased individuals. Serum samples from women diagnosed with epithelial ovarian cancer (EOC, <i>n</i> = 84) and matched healthy controls (<i>n</i> = 84) were obtained from the Gynecologic Oncology Group. Immunoglobulin (IgG, IgA, and IgM) concentrations and glycosylation profiles were quantified using multiple reaction monitoring mass spectrometry. Differential and classification analyses were performed to identify aberrant protein-specific glycopeptides using a training set. All findings were validated in an independent test set. Multiple glycopeptides from immunoglubins IgA, IgG, and IgM were found to be differentially expressed in serum of EOC patients compared with controls. The protein-specific glycosylation profiles showed their potential in the diagnosis of EOC. In particular, IgG-specific glycosylation profiles are the most powerful in discriminating between EOC case and controls. Additional studies of protein- and site-specific glycosylation profiles of immunoglobulins and other proteins will allow further elaboration on the characteristics of biological functionality and causality of the differential glycosylation in ovarian cancer and thus ultimately lead to increased sensitivity and specificity of diagnosis

Protein-Specific Differential Glycosylation of Immunoglobulins in Serum of Ovarian Cancer Patients

Previous studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian cancer; however, it was unclear to which proteins these glycans belong. We hypothesize that protein-specific glycosylation profiles of the glycans may be more informative of ovarian cancer and can provide insight into biological mechanisms underlying glycan aberration in serum of diseased individuals. Serum samples from women diagnosed with epithelial ovarian cancer (EOC, <i>n</i> = 84) and matched healthy controls (<i>n</i> = 84) were obtained from the Gynecologic Oncology Group. Immunoglobulin (IgG, IgA, and IgM) concentrations and glycosylation profiles were quantified using multiple reaction monitoring mass spectrometry. Differential and classification analyses were performed to identify aberrant protein-specific glycopeptides using a training set. All findings were validated in an independent test set. Multiple glycopeptides from immunoglubins IgA, IgG, and IgM were found to be differentially expressed in serum of EOC patients compared with controls. The protein-specific glycosylation profiles showed their potential in the diagnosis of EOC. In particular, IgG-specific glycosylation profiles are the most powerful in discriminating between EOC case and controls. Additional studies of protein- and site-specific glycosylation profiles of immunoglobulins and other proteins will allow further elaboration on the characteristics of biological functionality and causality of the differential glycosylation in ovarian cancer and thus ultimately lead to increased sensitivity and specificity of diagnosis

Multiple Reaction Monitoring for the Quantitation of Serum Protein Glycosylation Profiles: Application to Ovarian Cancer

Protein glycosylation fingerprints are widely recognized as potential markers for disease states, and indeed differential glycosylation has been identified in multiple types of autoimmune diseases and several types of cancer. However, releasing the glycans leave the glycoproteins unknown; therefore, there exists a need for high-throughput methods that allow quantification of site- and protein-specific glycosylation patterns from complex biological mixtures. In this study, a targeted multiple reaction monitoring (MRM)-based method for the protein- and site-specific quantitation involving serum proteins immunoglobulins A, G and M, alpha-1-antitrypsin, transferrin, alpha-2-macroglobulin, haptoglobin, alpha-1-acid glycoprotein and complement C3 was developed. The method is based on tryptic digestion of serum glycoproteins, followed by immediate reverse phase UPLC-QQQ-MS analysis of glycopeptides. To quantitate protein glycosylation independent of the protein serum concentration, a nonglycosylated peptide was also monitored. Using this strategy, 178 glycopeptides and 18 peptides from serum glycoproteins are analyzed with good repeatability (interday CVs of 3.65–21–92%) in a single 17 min run. To assess the potential of the method, protein glycosylation was analyzed in serum samples from ovarian cancer patients and controls. A training set consisting of 40 cases and 40 controls was analyzed, and differential analyses were performed to identify aberrant glycopeptide levels. All findings were validated in an independent test set (<i>n</i> = 44 cases and <i>n</i> = 44 controls). In addition to the differential glycosylation on the immunoglobulins, which was reported previously, aberrant glycosylation was also observed on each of the glycoproteins, which could be corroborated in the test set. This report shows the development of a method for targeted protein- and site-specific glycosylation analysis and the potential of such methods in biomarker development

Multiple Reaction Monitoring for the Quantitation of Serum Protein Glycosylation Profiles: Application to Ovarian Cancer

Protein glycosylation fingerprints are widely recognized as potential markers for disease states, and indeed differential glycosylation has been identified in multiple types of autoimmune diseases and several types of cancer. However, releasing the glycans leave the glycoproteins unknown; therefore, there exists a need for high-throughput methods that allow quantification of site- and protein-specific glycosylation patterns from complex biological mixtures. In this study, a targeted multiple reaction monitoring (MRM)-based method for the protein- and site-specific quantitation involving serum proteins immunoglobulins A, G and M, alpha-1-antitrypsin, transferrin, alpha-2-macroglobulin, haptoglobin, alpha-1-acid glycoprotein and complement C3 was developed. The method is based on tryptic digestion of serum glycoproteins, followed by immediate reverse phase UPLC-QQQ-MS analysis of glycopeptides. To quantitate protein glycosylation independent of the protein serum concentration, a nonglycosylated peptide was also monitored. Using this strategy, 178 glycopeptides and 18 peptides from serum glycoproteins are analyzed with good repeatability (interday CVs of 3.65–21–92%) in a single 17 min run. To assess the potential of the method, protein glycosylation was analyzed in serum samples from ovarian cancer patients and controls. A training set consisting of 40 cases and 40 controls was analyzed, and differential analyses were performed to identify aberrant glycopeptide levels. All findings were validated in an independent test set (<i>n</i> = 44 cases and <i>n</i> = 44 controls). In addition to the differential glycosylation on the immunoglobulins, which was reported previously, aberrant glycosylation was also observed on each of the glycoproteins, which could be corroborated in the test set. This report shows the development of a method for targeted protein- and site-specific glycosylation analysis and the potential of such methods in biomarker development

Multiple Reaction Monitoring for the Quantitation of Serum Protein Glycosylation Profiles: Application to Ovarian Cancer

Protein glycosylation fingerprints are widely recognized as potential markers for disease states, and indeed differential glycosylation has been identified in multiple types of autoimmune diseases and several types of cancer. However, releasing the glycans leave the glycoproteins unknown; therefore, there exists a need for high-throughput methods that allow quantification of site- and protein-specific glycosylation patterns from complex biological mixtures. In this study, a targeted multiple reaction monitoring (MRM)-based method for the protein- and site-specific quantitation involving serum proteins immunoglobulins A, G and M, alpha-1-antitrypsin, transferrin, alpha-2-macroglobulin, haptoglobin, alpha-1-acid glycoprotein and complement C3 was developed. The method is based on tryptic digestion of serum glycoproteins, followed by immediate reverse phase UPLC-QQQ-MS analysis of glycopeptides. To quantitate protein glycosylation independent of the protein serum concentration, a nonglycosylated peptide was also monitored. Using this strategy, 178 glycopeptides and 18 peptides from serum glycoproteins are analyzed with good repeatability (interday CVs of 3.65–21–92%) in a single 17 min run. To assess the potential of the method, protein glycosylation was analyzed in serum samples from ovarian cancer patients and controls. A training set consisting of 40 cases and 40 controls was analyzed, and differential analyses were performed to identify aberrant glycopeptide levels. All findings were validated in an independent test set (<i>n</i> = 44 cases and <i>n</i> = 44 controls). In addition to the differential glycosylation on the immunoglobulins, which was reported previously, aberrant glycosylation was also observed on each of the glycoproteins, which could be corroborated in the test set. This report shows the development of a method for targeted protein- and site-specific glycosylation analysis and the potential of such methods in biomarker development

Comprehensive Profiles of Human Milk Oligosaccharides Yield Highly Sensitive and Specific Markers for Determining Secretor Status in Lactating Mothers

Human milk oligosaccharides (HMOs), as an abundant and bioactive component of breast milk, work in many ways to promote the health of breast fed infants. The expression of HMOs has been shown to vary in accordance with Lewis blood type and secretor status, as women of different blood types differ in the expression of α1,2 fucosyltransferase (FUT2) and α1,3/4 fucosyltransferase (FUT3). In this study, HMOs were extracted from the milk of 60 women from The Gambia, Africa with various Lewis and secretor blood types. The HMOs were profiled using high resolution HPLC-Chip/TOF mass spectrometry. Notably, the amounts of fucosylation varied significantly between Le­(a+b-) nonsecretors, Le­(a-b+) and Le­(a-b-) secretors, and Le­(a-b-) nonsecretors. With higher frequency of expression of the recessive Lewis negative and nonsecretor phenotypes in West African populations, the HMO profiles of several milks from women of these phenotypes were examined, demonstrating decreased amounts of total oligosaccharide abundance and lower relative amounts of fucosylation. Also in this study, four specific fucosylated structures (2′FL, LNFP I, LDFT, and LNDFH I) were determined to be specific and sensitive glycan markers for rapidly determining secretor status without the need for serological testing

Differential N‑Glycosylation Patterns in Lung Adenocarcinoma Tissue

To decrease the mortality of lung cancer, better screening and diagnostic tools as well as treatment options are needed. Protein glycosylation is one of the major post-translational modifications that is altered in cancer, but it is not exactly clear which glycan structures are affected. A better understanding of the glycan structures that are differentially regulated in lung tumor tissue is highly desirable and will allow us to gain greater insight into the underlying biological mechanisms of aberrant glycosylation in lung cancer. Here, we assess differential glycosylation patterns of lung tumor tissue and nonmalignant tissue at the level of individual glycan structures using nLC–chip–TOF–MS. Using tissue samples from 42 lung adenocarcinoma patients, 29 differentially expressed (FDR < 0.05) glycan structures were identified. The levels of several oligomannose type glycans were upregulated in tumor tissue. Furthermore, levels of fully galactosylated glycans, some of which were of the hybrid type and mostly without fucose, were decreased in cancerous tissue, whereas levels of non- or low-galactosylated glycans mostly with fucose were increased. To further assess the regulation of the altered glycosylation, the glycomics data was compared to publicly available gene expression data from lung adenocarcinoma tissue compared to nonmalignant lung tissue. The results are consistent with the possibility that the observed N-glycan changes have their origin in differentially expressed glycosyltransferases. These results will be used as a starting point for the further development of clinical glycan applications in the fields of imaging, drug targeting, and biomarkers for lung cancer