Mapping the O‐glycoproteome using site‐specific extraction of O‐linked glycopeptides (EXoO)

Abstract

Protein glycosylation is one of the most abundant post‐translational modifications. However, detailed analysis of O‐linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of suitable methodologies. Here, a chemoenzymatic method is introduced for the site‐specific extraction of O‐linked glycopeptides (EXoO), which enabled the mapping of over 3,000 O‐linked glycosylation sites and definition of their glycans on over 1,000 proteins in human kidney tissues, T cells, and serum. This large‐scale localization of O‐linked glycosylation sites demonstrated that EXoO is an effective method for defining the site‐specific O‐linked glycoproteome in different types of sample. Detailed structural analysis of the sites identified revealed conserved motifs and topological orientations facing extracellular space, the cell surface, the lumen of the Golgi, and the endoplasmic reticulum (ER). EXoO was also able to reveal significant differences in the O‐linked glycoproteome of tumor and normal kidney tissues pointing to its broader use in clinical diagnostics and therapeutics.

EXoO-Tn: Tag-n-Map the Tn Antigen in the Human Proteome

“…Also, the identification of glycopeptides with two or three Gal( 13 C 6 )-Tn compositions suggests many more glycosylation sites in the peptide sequences supporting an even larger number of Tn-glycosylation sites in Jurkat cells. Characterization of glycosylation sites and glycoproteins identified in Jurkat cells revealed conserved features of protein O-linked glycosylation, including consensus motif, cellular localization, and distribution of the relative position of glycosylation sites across the protein sequences, a reminiscence of that seen in human kidney, serum, and T cells in the previous study 21 . Given that Tn is prevalent in cancers and other diseases, EXoO-Tn is anticipated to have broad translational and clinical utilities.…”

“…Trypsin was added to the samples with an enzyme/protein ratio of 1/40 w/w. After incubation at 37°C for 16 hours, lysine residues were guanidination-modified, and peptides were desalted using C18 cartridges (Waters, Milford, MA), as described in the previous study 21 . The peptides were dried using speed-vac, resuspended in PBS with α2-3,6,8 neuraminidase (New England Biolabs, Ipswich, MA), and incubated at 37°C for 16 hours.…”

“…Subsequent analysis using HCD-MS2 identifies 96 O-glycoproteins in three experiments with 87 glycosylation sites being localized in the first experiment of Jurkat cells 20 . We, however, anticipate that about a thousand Tn-glycosylation sites remain to be mapped in Jurkat cells because 1,295 O-linked glycosylation sites are mapped in CEM cells, a human T cell line, using a method named EXoO developed in previous study 21 . It appears that the development of a technology capable of large-scale mapping of Tn-glycosylation sites would be a significant advance in technology and cancer biology.…”

Separation and detection of minimal length glycopeptide neoantigen epitopes centering the GSTA region of MUC1 by liquid chromatography/mass spectrometry

“…Significant progress has been made for glycopeptide analysis in the past decade. Databases and methods for analyzing N‐glycopeptides and O‐glycopeptides are accumulating, especially for site‐specific glycosylation. Obviously, data containing both identities of glycan structures and glycosylation sites are most valuable for their functional analysis, compared with data containing glycan structures or glycosylation sites alone.…”

“…The group of Levery and Clausen published systemic analysis of O‐glycopeptides in trypsin‐digested proteins from CHO cells . Zhang and co‐workers recently published site‐specific extraction of O‐linked glycopeptides in trypsin‐digested tissues . However, no data has been available for the TR region of mucin‐1 protein yet due to the resistance of the TR region to trypsin digestion.…”