The study of glycosylation patterns (glycomics) in biological samples is an emerging field that can provide key insights into cell development and pathology. A current challenge in the field of glycomics is to determine how to quantify changes in glycan expression between different cells, tissues, or biological fluids. Here we describe a novel strategy, quantitation by isobaric labeling (QUIBL), to facilitate comparative glycomics. Permethylation of a glycan with 13CH3I or 12CH2DI generates a pair of isobaric derivatives, which have the same nominal mass. However, each methylation site introduces a mass difference of 0.002922 Da. As glycans have multiple methylation sites, the total mass difference for the isobaric pair allows separation and quantitation at a resolution of ∼30000 m/Δm. N-Linked oligosaccharides from a standard glycoprotein and human serum were used to demonstrate that QUIBL facilitates relative quantitation over a linear dynamic range of 2 orders of magnitude and permits the relative quantitation of isomeric glycans. We applied QUIBL to quantitate glycomic changes associated with the differentiation of murine embryonic stem cells to embryoid bodies.Keywords: glycomics; isobaric labeling; quantitation; stable isotope labeling;

Robust quantification is an essential component of comparative -omic strategies. In this regard, glycomics lags behind proteomics. Although various isotope-tagging and direct quantification methods have recently enhanced comparative glycan analysis, a cell culture labeling strategy, that could provide for glycomics the advantages that SILAC provides for proteomics, has not been described. Here, we report the development of IDAWG, Isotopic Detection of Aminosugars With Glutamine, for the incorporation of differential mass tags into the glycans of cultured cells. In this method, culture media containing amide-15N-Gln is used to metabolically label cellular aminosugars with heavy nitrogen. Because the amide side chain of Gln is the sole source of nitrogen for the biosynthesis of GlcNAc, GalNAc, and sialic acid, we demonstrate that culturing mouse embryonic stems cells for 72 h in the presence of amide-15N-Gln media results in nearly complete incorporation of 15N into N-linked and O-linked glycans. The isotopically heavy monosaccharide residues provide additional information for interpreting glycan fragmentation and also allow quantification in both full MS and MS/MS modes. Thus, IDAWG is a simple to implement, yet powerful quantitative tool for the glycomics toolbox.Keywords: Glycosylation; IDAWG; Mass Spectrometry; Metabolic labeling; Quantitative glycomics;

Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein’s function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications.

The study of N-linked glycans is among the most challenging bioanalytical tasks because of their complexity and variety. The presence of glycoform families that differ only in branching and/or linkage position makes the identification and quantitation of individual glycans exceedingly difficult. Quantitation of these individual glycans is important because changes in the abundance of these isomers are often associated with significant biomedical events. For instance, previous studies have shown that the ratio of α2-3 to α2-6 linked sialic acid (SA) plays an important role in cancer biology. Consequently, quantitative methods to detect alterations in the ratios of glycans based on their SA linkages could serve as a diagnostic tool in oncology, yet traditional glycomic profiling cannot readily differentiate between these linkage isomers. Here, we present a liquid chromatography-selected reaction monitoring (LC-SRM) approach that we demonstrate is capable of quantitating the individual SA linkage isomers. The LC method is capable of separating sialylated N-glycan isomers differing in α2-3 and α2-6 linkages using a novel superficially porous particle (Fused-Core) Penta-HILIC (hydrophilic interaction liquid chromatography) column. SRM detection provides the relative quantitation of each SA linkage isomer, and minimizes interferences from coeluting glycans that are problematic for UV/Fluorescence based quantitation. With our approach, the relative quantitation of each SA linkage isomer is obtained from a straightforward liquid chromatography-mass spectrometry (LC-MS) experiment.

Botrytis cinerea (B. cinerea) is a filamentous fungus infecting more than 200 plant species, causing significant economic losses worldwide. Secreted proteins are released as an initial response of the fungus to its plant host. We report the use of a high-throughput LC-MS/MS approach to analyze B. cinerea BO5.10 secreted proteins. Secretions were collected from fungus grown on a solid substrate of cellophane membrane while mock infecting media supplemented with the extract of full red tomato, ripened strawberry or Arabidopsis leaf extract. Overall, 89 B. cinerea proteins were identified from all growth conditions. Sixty proteins were predicted to contain a SignalP motif indicating the extracellular location of the proteins. Seven proteins were observed in all the growth conditions implying a constitutive nature of their secretion. Identified in the secretions were transport proteins, proteins well-characterized for carbohydrate metabolism, peptidases, oxidation/reduction, and pathogenicity factors that provide important insights into how B. cinerea may use secreted proteins for plant infection and colonization.

The study of glycosylation patterns (glycomics) in biological samples is an emerging field that can provide key insights into cell development and pathology. A current challenge in the field of glycomics is to determine how to quantify changes in glycan expression between different cells, tissues, or biological fluids. Here we describe a novel strategy, quantitation by isobaric labeling (QUIBL), to facilitate comparative glycomics. Permethylation of a glycan with 13CH 3I or 12CH 2DI generates a pair of isobaric derivatives, which have the same nominal mass. However, each methylation site introduces a mass difference of 0.002922 Da. As glycans have multiple methylation sites, the total mass difference for the isobaric pair allows separation and quantitation at a resolution of ∼30000 m/Δ m. N-Linked oligosaccharides from a standard glycoprotein and human serum were used to demonstrate that QUIBL facilitates relative quantitation over a linear dynamic range of 2 orders of magnitude and permits the relative quantitation of isomeric glycans. We applied QUIBL to quantitate glycomic changes associated with the differentiation of murine embryonic stem cells to embryoid bodies.