Enhanced Detection and Identification of Glycopeptides in Negative Ion Mode Mass Spectrometry

A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS) in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode MS analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collision-induced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positive- and negative ion modes after digestion with bead-immobilized Pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved site-specific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed toward enhanced N- and O-linked glycopeptide detection and to serve as a complementary tool to positive ion mode MS/MS analysis

Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ): Application to Glycomics

A new relative quantification strategy for glycomics, named deuterium oxide (D2O) labeling for global omics relative quantification (DOLGOReQ), has been developed based on the partial metabolic D2O labeling, which induces a subtle change in the isotopic distribution of glycan ions. The relative abundance of unlabeled to D-labeled glycans was extracted from the overlapped isotopic envelope obtained from a mixture containing equal amounts of unlabeled and D-labeled glycans. The glycan quantification accuracy of DOLGOReQ was examined with mixtures of unlabeled and D-labeled HeLa glycans combined in varying ratios according to the number of cells present in the samples. The relative quantification of the glycans mixed in an equimolar ratio revealed that 92.4 and 97.8% of the DOLGOReQ results were within a 1.5- and 2-fold range of the predicted mixing ratio, respectively. Furthermore, the dynamic quantification range of DOLGOReQ was investigated with unlabeled and D-labeled HeLa glycans mixed in different ratios from 20:1 to 1:20. A good correlation (Pearson’s r > 0.90) between the expected and measured quantification ratios over 2 orders of magnitude was observed for 87% of the quantified glycans. DOLGOReQ was also applied in the measurement of quantitative HeLa cell glycan changes that occur under normoxic and hypoxic conditions. Given that metabolic D2O labeling can incorporate D into all types of glycans, DOLGOReQ has the potential as a universal quantification platform for large-scale comparative glycomic experiments

Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ): Application to Glycomics

A new relative quantification strategy for glycomics, named deuterium oxide (D2O) labeling for global omics relative quantification (DOLGOReQ), has been developed based on the partial metabolic D2O labeling, which induces a subtle change in the isotopic distribution of glycan ions. The relative abundance of unlabeled to D-labeled glycans was extracted from the overlapped isotopic envelope obtained from a mixture containing equal amounts of unlabeled and D-labeled glycans. The glycan quantification accuracy of DOLGOReQ was examined with mixtures of unlabeled and D-labeled HeLa glycans combined in varying ratios according to the number of cells present in the samples. The relative quantification of the glycans mixed in an equimolar ratio revealed that 92.4 and 97.8% of the DOLGOReQ results were within a 1.5- and 2-fold range of the predicted mixing ratio, respectively. Furthermore, the dynamic quantification range of DOLGOReQ was investigated with unlabeled and D-labeled HeLa glycans mixed in different ratios from 20:1 to 1:20. A good correlation (Pearson’s r > 0.90) between the expected and measured quantification ratios over 2 orders of magnitude was observed for 87% of the quantified glycans. DOLGOReQ was also applied in the measurement of quantitative HeLa cell glycan changes that occur under normoxic and hypoxic conditions. Given that metabolic D2O labeling can incorporate D into all types of glycans, DOLGOReQ has the potential as a universal quantification platform for large-scale comparative glycomic experiments

Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ): Application to Glycomics

A new relative quantification strategy for glycomics, named deuterium oxide (D2O) labeling for global omics relative quantification (DOLGOReQ), has been developed based on the partial metabolic D2O labeling, which induces a subtle change in the isotopic distribution of glycan ions. The relative abundance of unlabeled to D-labeled glycans was extracted from the overlapped isotopic envelope obtained from a mixture containing equal amounts of unlabeled and D-labeled glycans. The glycan quantification accuracy of DOLGOReQ was examined with mixtures of unlabeled and D-labeled HeLa glycans combined in varying ratios according to the number of cells present in the samples. The relative quantification of the glycans mixed in an equimolar ratio revealed that 92.4 and 97.8% of the DOLGOReQ results were within a 1.5- and 2-fold range of the predicted mixing ratio, respectively. Furthermore, the dynamic quantification range of DOLGOReQ was investigated with unlabeled and D-labeled HeLa glycans mixed in different ratios from 20:1 to 1:20. A good correlation (Pearson’s r > 0.90) between the expected and measured quantification ratios over 2 orders of magnitude was observed for 87% of the quantified glycans. DOLGOReQ was also applied in the measurement of quantitative HeLa cell glycan changes that occur under normoxic and hypoxic conditions. Given that metabolic D2O labeling can incorporate D into all types of glycans, DOLGOReQ has the potential as a universal quantification platform for large-scale comparative glycomic experiments

Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ): Application to Glycomics

A new relative quantification strategy for glycomics, named deuterium oxide (D2O) labeling for global omics relative quantification (DOLGOReQ), has been developed based on the partial metabolic D2O labeling, which induces a subtle change in the isotopic distribution of glycan ions. The relative abundance of unlabeled to D-labeled glycans was extracted from the overlapped isotopic envelope obtained from a mixture containing equal amounts of unlabeled and D-labeled glycans. The glycan quantification accuracy of DOLGOReQ was examined with mixtures of unlabeled and D-labeled HeLa glycans combined in varying ratios according to the number of cells present in the samples. The relative quantification of the glycans mixed in an equimolar ratio revealed that 92.4 and 97.8% of the DOLGOReQ results were within a 1.5- and 2-fold range of the predicted mixing ratio, respectively. Furthermore, the dynamic quantification range of DOLGOReQ was investigated with unlabeled and D-labeled HeLa glycans mixed in different ratios from 20:1 to 1:20. A good correlation (Pearson’s r > 0.90) between the expected and measured quantification ratios over 2 orders of magnitude was observed for 87% of the quantified glycans. DOLGOReQ was also applied in the measurement of quantitative HeLa cell glycan changes that occur under normoxic and hypoxic conditions. Given that metabolic D2O labeling can incorporate D into all types of glycans, DOLGOReQ has the potential as a universal quantification platform for large-scale comparative glycomic experiments

Annotation of a Serum N-Glycan Library for Rapid Identification of Structures

Glycosylation is one of the most common post-translational modifications of proteins and has been shown to change with various pathological states including cancer. Global glycan profiling of human serum based on mass spectrometry has already led to several promising markers for diseases. The changes in glycan structure can result in altered monosaccharide composition as well as in the linkages between the monosaccharides. High-throughput glycan structural elucidation is not possible because of the lack of a glycan template to expedite identification. In an effort toward rapid profiling and identification of glycans, we have constructed a library of structures for the serum glycome to aid in the rapid identification of serum glycans. N-Glycans from human serum glycoproteins are used as a standard and compiled into a library with exact structure (composition and linkage), liquid chromatography retention time, and accurate mass. Development of the library relies on highly reproducible nanoLC–MS retention times. Tandem MS and exoglycosidase digestions were used for structural elucidation. The library currently contains over 300 entries with 50 structures completely elucidated and over 60 partially elucidated structures. This database is steadily growing and will be used to rapidly identify glycans in unknown biological samples

Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ): Application to Glycomics

A new relative quantification strategy for glycomics, named deuterium oxide (D2O) labeling for global omics relative quantification (DOLGOReQ), has been developed based on the partial metabolic D2O labeling, which induces a subtle change in the isotopic distribution of glycan ions. The relative abundance of unlabeled to D-labeled glycans was extracted from the overlapped isotopic envelope obtained from a mixture containing equal amounts of unlabeled and D-labeled glycans. The glycan quantification accuracy of DOLGOReQ was examined with mixtures of unlabeled and D-labeled HeLa glycans combined in varying ratios according to the number of cells present in the samples. The relative quantification of the glycans mixed in an equimolar ratio revealed that 92.4 and 97.8% of the DOLGOReQ results were within a 1.5- and 2-fold range of the predicted mixing ratio, respectively. Furthermore, the dynamic quantification range of DOLGOReQ was investigated with unlabeled and D-labeled HeLa glycans mixed in different ratios from 20:1 to 1:20. A good correlation (Pearson’s r > 0.90) between the expected and measured quantification ratios over 2 orders of magnitude was observed for 87% of the quantified glycans. DOLGOReQ was also applied in the measurement of quantitative HeLa cell glycan changes that occur under normoxic and hypoxic conditions. Given that metabolic D2O labeling can incorporate D into all types of glycans, DOLGOReQ has the potential as a universal quantification platform for large-scale comparative glycomic experiments

The expression of proteins in HMO cluster in <i>B. infantis</i> during growth on different prebiotics.

<p>(a) Graphical display of genes in HMO cluster. Proteins involved in the HMO translocation and degradation are noted with the locus tag under the gene. Sets of genes inside the red box are the transport systems that comprise a Family 1 extracellular SBP and two inner-membrane components. Bars represent the relative amounts of each protein in different carbon sources. Black and gray bars indicate the NSAF values in the soluble and insoluble fraction, respectively. (b)∼(e) are the glycosyl hydrolases for HMO degradation. (f)∼(k) are the Family 1 SBPs and (l)∼(n) are the proteins whose the amount was high but the role in HMO metabolism is not well known.</p