Mapping site-specific protein N-glycosylations through liquid chromatography/mass spectrometry and targeted tandem mass spectrometry.
Glycosylation is one of the most common posttranslational modifications (PTMs) of proteins, the characterization of which is commonly achieved through proteomic protocol, involving trypsin digestion followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, it is often not possible to characterize all glycopeptides in a complex sample because of the high complexity of glycoproteomic samples, and the relative lower abundances of glycopeptides in comparison to the unmodified peptides. We present here a targeted MS/MS analysis approach, which utilizes a previously developed computational tool, GlyPID, to guide multiple experiments, thus permitting a complete characterization of all N-glycosylation sites of glycoproteins present in a complex sample. We have tested our approach using model glycoproteins analyzed by high-resolution LTQ-FT MS. The results demonstrate a potential use of our method for a high-throughput characterization of complex mixtures of glycosylated proteins.
Wu
Yin
Y
School of Informatics and Computing, Indiana University, Bloomington, IN 47408, USA.
Mechref
Yehia
Y
Klouckova
Iveta
I
Mayampurath
Anoop
A
Novotny
Milos V
MV
Tang
Haixu
H
D000465:Algorithms; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D045424:Complex Mixtures / Q000737:chemistry; D019295:Computational Biology / Q000379:methods*; D006020:Glycopeptides / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D010449:Peptide Mapping / Q000379:methods*; D011499:Protein Processing, Post-Translational; D015203:Reproducibility of Results; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000509:alpha-Fetoproteins / Q000737:chemistry
D045424:Complex Mixtures; D006020:Glycopeptides; D000509:alpha-Fetoproteins