Glycoprotein disease markers and single protein-omics

Abstract

Glycoproteins are well represented among biomarkers for inflammatory and cancer diseases. Secreted and membrane-associated glycoproteins make excellent targets for noninvasive detection. In this review, we discuss clinically applicable markers of cancer diseases and methods for their analysis. High throughput discovery continues to supply marker candidates with unusual glycan structures, altered glycoprotein abundance, or distribution of site-specific glycoforms. Improved analytical methods are needed to unlock the potential of these discoveries in validated clinical assays. A new generation of targeted quantitative assays is expected to advance the use of glycoproteins in early detection of diseases, molecular disease classification, and monitoring of therapeutic interventions.

Fig. 1.

Glycan structures associated with cancer diseases. A, four truncated O-glycan structures; B, representative N-glycan linkages (circled); and C, four outer arm fucosylated structures that are commonly found in cancer diseases together with their sialylated forms. Tn, GalNAcα1-O-Ser/Thr; S-Tn, Neu5Acα2–6GalNAcα1-O-Ser/Thr; T, Galβ1–3-GalNAcα-O-Ser/Thr; S-T, Neu5Acα2–3-Galβ1–3-GalNAcα-O-Ser/Thr.

Fig. 2.

Schematic of glycoforms and proteolytic fragments of UniProtKB/SwissProt entry Q14624. A, full-length protein with signal peptide, domains (labeled), N-glycosylation sites (black circles), and proposed O-glycosylation sites (white circles); B, cleavage of mature protein by plasma kallikrein; C, additional internal cleavage of protein fragments. Asterisks indicate proteolytic fragments (frag.) detected in serum.

Fig. 3.

Toward quantitative measurement of site-specific glycoforms of proteins. A, one strategy for quantitative measurement of glycopeptides is LC-MS-MRM. Optimized quantification of appropriate oxonium ions, peptide-glycan fragments, or peptide b and y ions is expected to facilitate detection of specific types of glycopeptides. B, schematic of N-glycopeptide fragmentation. C, schematic of O-glycopeptide fragmentation. D, spectrum of N-glycopeptide LPTQNITFQTE-HexNAc4Hex5NeuAc1Fuc1 fragmented via CID. E, spectrum of O-glycopeptide ASFSPR-HexNAcHexNeuAc2 fragmented using CID.