TY - JOUR
T1 - Characterization of sialylated and fucosylated glycopeptides of b2-glycoprotein i by a combination of HILIC LC and MALDI MS/MS
AU - Kondo, Akira
AU - Thaysen-Andersen, Morten
AU - Hjernø, Karin
AU - Jensen, Ole N.
PY - 2010
Y1 - 2010
N2 - Characterization of low microgram levels of glycoprotein remains a challenge due to extensive heterogeneity of the conjugated N-glycans at each individual glycosylation site. We present an optimized, sensitive workflow for glycopeptide isolation and characterization that exploits the complementary features of RP (Poros R2) and hydrophilic (zwitter-ionic hydrophilic interaction chromatography) chromatographic resins. The glycopeptide analysis workflow was applied to human β2-glycoprotein I (β2-GPI, apolipoprotein H), which contains multiple N-glycosylation sites. Conditions for rapid proteolytic digestion of β2-GPI using low-specificity proteases were optimized to detect β2-GPI glycopeptides by MS. We demonstrate the importance of ensuring sufficient column capacity of both hydrophobic and hydrophilic stationary phases for optimal glycoprofiling by MS. The enriched glycopeptides were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi- and triantennary complex type glycans, were characterized at three N-glycosylation sites, namely Asn-143, Asn-174 and Asn-234, of β2-GPI. Further exploration of the complementary nature of RP and HILIC stationary phases for glycopeptide isolation prior to MS analysis may eventually enable systematic analysis of complex glycoprotein samples in functional proteomic research and advance our understanding of the biological role of protein glycosylation.
AB - Characterization of low microgram levels of glycoprotein remains a challenge due to extensive heterogeneity of the conjugated N-glycans at each individual glycosylation site. We present an optimized, sensitive workflow for glycopeptide isolation and characterization that exploits the complementary features of RP (Poros R2) and hydrophilic (zwitter-ionic hydrophilic interaction chromatography) chromatographic resins. The glycopeptide analysis workflow was applied to human β2-glycoprotein I (β2-GPI, apolipoprotein H), which contains multiple N-glycosylation sites. Conditions for rapid proteolytic digestion of β2-GPI using low-specificity proteases were optimized to detect β2-GPI glycopeptides by MS. We demonstrate the importance of ensuring sufficient column capacity of both hydrophobic and hydrophilic stationary phases for optimal glycoprofiling by MS. The enriched glycopeptides were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi- and triantennary complex type glycans, were characterized at three N-glycosylation sites, namely Asn-143, Asn-174 and Asn-234, of β2-GPI. Further exploration of the complementary nature of RP and HILIC stationary phases for glycopeptide isolation prior to MS analysis may eventually enable systematic analysis of complex glycoprotein samples in functional proteomic research and advance our understanding of the biological role of protein glycosylation.
UR - http://www.scopus.com/inward/record.url?scp=77950796589&partnerID=8YFLogxK
U2 - 10.1002/jssc.200900802
DO - 10.1002/jssc.200900802
M3 - Article
C2 - 20209506
AN - SCOPUS:77950796589
SN - 1615-9306
VL - 33
SP - 891
EP - 902
JO - Journal of Separation Science
JF - Journal of Separation Science
IS - 6-7
ER -