TY - JOUR
T1 - Assigning glycosylation sites and microheterogeneities in glycoproteins by liquid chromatography/tandem mass spectrometry.
AU - Mechref, Yehia
AU - Madera, Milan
AU - Novotny, Milos V.
PY - 2009
Y1 - 2009
N2 - Glycosylation of proteins is one of the most common posttranslational modifications which has its bearing on function and biological activity. Assigning the glycosylation sites and their inherent microheterogeneities are key structural issues addressing various glycoprotein functions. This chapter describes three different approaches all based on liquid chromatography/tandem mass spectrometry (LC/MS-MS), which are commonly employed for the assignment of protein glycosylation sites and their microheterogeneities. Comparing the LC/MS-MS analysis of a native glycoprotein tryptic digest to that of a deglycosylated tryptic digest can be accomplished through a routine LC/MS instrument. The use of a scanning mass spectrometer capable of switching between high-voltage and low-voltage scans, combined with monitoring carbohydrate-characteristic oxonium ions, is yet another analytical approach utilized for characterization of the glycosylation sites of glycoproteins. These two approaches do not address the problem originating from the ion suppression associated with coeluting peptides. The use of on-line glycopeptide enrichment in conjunction with LC/MS-MS is a third approach, which reduces ion suppression, thus offering a more sensitive approach to the characterization of protein glycosylation sites.
AB - Glycosylation of proteins is one of the most common posttranslational modifications which has its bearing on function and biological activity. Assigning the glycosylation sites and their inherent microheterogeneities are key structural issues addressing various glycoprotein functions. This chapter describes three different approaches all based on liquid chromatography/tandem mass spectrometry (LC/MS-MS), which are commonly employed for the assignment of protein glycosylation sites and their microheterogeneities. Comparing the LC/MS-MS analysis of a native glycoprotein tryptic digest to that of a deglycosylated tryptic digest can be accomplished through a routine LC/MS instrument. The use of a scanning mass spectrometer capable of switching between high-voltage and low-voltage scans, combined with monitoring carbohydrate-characteristic oxonium ions, is yet another analytical approach utilized for characterization of the glycosylation sites of glycoproteins. These two approaches do not address the problem originating from the ion suppression associated with coeluting peptides. The use of on-line glycopeptide enrichment in conjunction with LC/MS-MS is a third approach, which reduces ion suppression, thus offering a more sensitive approach to the characterization of protein glycosylation sites.
UR - http://www.scopus.com/inward/record.url?scp=62349117587&partnerID=8YFLogxK
U2 - 10.1007/978-1-59745-493-3_9
DO - 10.1007/978-1-59745-493-3_9
M3 - Article
C2 - 19241032
AN - SCOPUS:62349117587
SN - 1064-3745
VL - 492
SP - 161
EP - 180
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -