Tandem 18O stable isotope labeling for quantification of N-glycoproteome.
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.
Liu
Ze
Z
Department of Chemistry, Fudan University, Shanghai, China.
Cao
Jing
J
He
Yifeng
Y
Qiao
Liang
L
Xu
Congjian
C
Lu
Haojie
H
Yang
Pengyuan
P
D000368:Aged; D005260:Female; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D010051:Ovarian Neoplasms / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D010103:Oxygen Isotopes / Q000737:chemistry; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results
D006023:Glycoproteins; D010103:Oxygen Isotopes; D020543:Proteome