Combined phospho- and glycoproteome enrichment in nephrocalcinosis tissues of phytate-fed rats.
BACKGROUND
Protein post-translational modifications (PTMs) are directly involved in protein function and cellular activities. Among them, glycosylation and phosphorylation are particularly important modifications on proteins located at extracellular and intracellular domains, respectively. However, the combined detection using phospho- and glycoproteomics is limited mainly due to protocol differences.


METHODS
In this study, we developed a novel method for both phospho- and glycoproteome detection from a single sample batch, in which a titanium dioxide cartridge was used to capture the phosphoproteome, and the flow-through solution was processed for capturing N-linked glycopeptides using hydrazide resin.


RESULTS
By using 1 mg of protein from kidney tissue lysates from normal and diseased rats, we concurrently identified 437 glycosites/358 phosphosites and 468 glycosites/369 phosphosites in normal and disease kidneys, respectively, by liquid chromatography/tandem mass spectrometric analysis.


CONCLUSIONS
Compared with individual PTM analyses, the combined PTM analysis clearly provides more broad implications for PTMs related to the pathological status and discovery of biomarker candidates. Furthermore, the combined protocol thoroughly showed its advantages in enrichment efficiency and biological interpretation compared with current methods.
Lee Gil Ya Cancer and Diabetes Institute, Gachon University, 7-45 Songdo-dong, Yeonsu-ku, Incheon, 406-840, Republic of Korea.
TrangHuyen
T
Tran
Jong-Moon
JM
Park
Ok-Hee
OH
Kim
Bora
B
Kim
Do-young
DY
Choi
JeongHwa
J
Lee
KwangPyo
K
Kim
Byung-Chul
BC
Oh
Hookeun
H
Lee
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D005260:Female; D006020:Glycopeptides / Q000032:analysis*; D007668:Kidney / Q000737:chemistry / Q000187:drug effects; D008969:Molecular Sequence Data; D009397:Nephrocalcinosis / Q000139:chemically induced* / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis*; D010833:Phytic Acid / Q000008:administration & dosage / Q000633:toxicity*; D020543:Proteome / Q000187:drug effects*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry
D006020:Glycopeptides; D010748:Phosphopeptides; D020543:Proteome; D010833:Phytic Acid