Targeted methods for quantitative analysis of protein glycosylation

Abstract

Quantification of proteins by LC-MS/MS-MRM has become a standard method with broad projected clinical applicability. MRM quantification of protein modifications is, however, far less utilized, especially in the case of glycoproteins. This review summarizes current methods for quantitative analysis of protein glycosylation with a focus on MRM methods. We describe advantages of this quantitative approach, analytical parameters that need to be optimized to achieve reliable measurements, and point out the limitations. Differences between major classes of N- and O-glycopeptides are described and class-specific glycopeptide assays are demonstrated.

Changes in the expression of N- and O-glycopeptides in patients with colorectal cancer and hepatocellular carcinoma quantified by full-MS scan FT-ICR and multiple reaction monitoring

“…Collision induced dissociation of glycopeptides (CID) yields high intensity oxonium ions that originate from the glycans and less intense peptide-glycan or peptide fragments. The highly sensitive but less specific oxonium ions are typically used as quantitative transitions in the MRM mode [13,25,47]. In complex sample mixture, the most intense peptide-glycan fragments are often selected as specific transitions used for glycopeptide confirmation.…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…Interesting developments have been described in targeted glycopeptide detection and quantitation strategies, as recently reviewed (139). Several applications of multiple reaction monitoring (MRM)-based glycopeptide analysis from a variety of biological systems and diseases were lately published including the use of MRM-based serum glycoproteomics to study esophagus diseases (140), liver disease (141), and the immunoglobulin subclasses (134,142,143).…”

Data Independent Analysis of IgG Glycoforms in Samples of Unfractionated Human Plasma

“…Limitations of these methods in the resolution of isobaric glycan structures or glycoforms of the IgG1-4 subclasses were described and are generally considered acceptable. 13–15 Analysis of IgG in the context of diseases is complicated by the variable background of the biological samples which limits, to some degree, the ability of the methods to quantify comprehensively the distribution of glycoforms. 15 It is therefore most common to analyze IgG isolated from the biological samples, typically by affinity enrichment on protein A or G resins.…”

“…13–15 Analysis of IgG in the context of diseases is complicated by the variable background of the biological samples which limits, to some degree, the ability of the methods to quantify comprehensively the distribution of glycoforms. 15 It is therefore most common to analyze IgG isolated from the biological samples, typically by affinity enrichment on protein A or G resins. 13,16 This enrichment step enables the use of the methods developed for the analysis of therapeutic IgG provided that sufficient amounts of representative IgG glycoforms are accessible.…”

“…14,19 The available MS methods are based most frequently on the quantification of precursor ion intensities 13,20 because the signal, especially in the case of isolated IgG, is typically sufficient for adequate coverage of the glycoform precursors. 14,21 Several groups reported also MRM quantification of oxonium ions generated by collision induced dissociation (CID) of glycopeptides as reviewed in ref 15. These common glycan fragments ( m / z 138, 204, 366) have high sensitivity but low specificity because they are generated from virtually all N-glycopeptides and have interferences from some peptide fragments as well.…”