Tandem ¹⁸O Stable Isotope Labeling for Quantification of N-Glycoproteome

Abstract

A new strategy using tandem (18)O stable isotope labeling (TOSIL) to quantify the N-glycosylation site occupancy is reported. Three heavy oxygen atoms are introduced into N-glycosylated peptides: two (18)O atoms are incorporated into the carboxyl terminal of all peptides during a tryptic digestion, and the third (18)O atom is incorporated into the N-glycosylation site of asparagines-linked sugar chains specifically via a N-glycosidase F (PNGase F)-mediated hydrolysis. Comparing samples treated in H(2)(18)O and samples treated in H(2)(16)O, a unique mass shift of 6 Da can be shown for N-glycosylated peptide with single glycosylation site, which could be easily distinguished from those nonglycosite peptide pairs with a mass difference of 4 Da only. The relative quantities of N-glycosylated and its parent protein-levels were obtained simultaneous by measuring the intensity ratios of (18)O/(16)O for glycosylated (6 Da) and for nonglycosylated (4 Da) peptides, respectively. Thus, a comparison of these two ratios can be utilized to evaluate the changes of occupancy of N-glycosylation at specific sites between healthy and diseased individuals. The TOSIL approach yielded good linearity in quantitative response within 10-fold dynamic range with the correlation coefficient r(2) > 0.99. The standard deviation (SD) ranged from 0.06 to 0.21, for four glycopeptides from two model glycoproteins. Furthermore, serums from a patient with ovarian cancer and healthy individual were used as test examples to validate the novel TOSIL method. A total of 86 N-glycosylation sites were quantified and N-glycosylation levels of 56 glycopeptides showed significant changes. Most changes in N-glycosylation at specific sites have the same trends as those of protein expression levels; however, the occupancies of three N-glycosylation sites were significantly changed with no change in proteins levels.

Site-specific Glycoforms of Haptoglobin in Liver Cirrhosis and Hepatocellular Carcinoma

“…Determination of Glyco-site Occupancy-Occupancies of all four Hp glycosites were quantified by comparison of XIC precursor ion intensities of unoccupied N-(asparagine) peptides and deglycosylated D-(aspartic acid) peptides following PNGaseF de-glycosylation under [ 18 O]water as described previously (30,31). Briefly, Hp (2 pmol) was digested after reduction and alkylation by trypsin or endoproteinase Glu-C as described above.…”

Mass Spectrometry Based Glycoproteomics—From a Proteomics Perspective

“…Attempts have been made to apply this approach to identify differentially expressed N-glycosylation associated with ovarian cancer in serum (207). In a different approach, the SILAC technique allows incorporation of stable isotope-labeled amino acids into proteins during cell culturing process (196), and was applied to investigate the difference in cell surface N-glycoproteins among different cell types (64).…”

The GlycoFilter: A Simple and Comprehensive Sample Preparation Platform for Proteomics, N-Glycomics and Glycosylation Site Assignment

“…Accordingly, many have reported that changes or variations in protein expression and glycosylation occupancy may not always be analogous or parallel (41,42). Even though a recent approach proposed analyzing the change of sitespecific occupancy between samples using only hydrazide enriched deglycopeptides (43), without a corresponding measurement of the sample specific protein expression this technique may result in incomplete measurements.…”