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Site‐specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion
Author(s) -
Schindler Patrick A.,
Settineri Christine A.,
Collet Xavier,
Fielding Christopher J.,
Burlingame Alma L.
Publication year - 1995
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560040419
Subject(s) - chemistry , glycopeptide , glycosylation , biochemistry , electrospray ionization , acyltransferase , chromatography , glycoprotein , glycan , mass spectrometry , enzyme , antibiotics
Site‐specific structural characterization of the glycosylation of human lecithinrcholesterol acyltransferase (LCAT) was carried out using microbore reversed‐phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate‐specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected TV‐linked glycopeptides of LCAT, a di‐ O ‐linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N ‐linked glycosylation sites (Asn 20, Asn 84, Asn 272, and Asn 384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O ‐linked glycosylation sites were identified at Thr 407 and Ser 409 of the LCAT O ‐linked glycopeptide, each of which contain sialylated galactoseβ13 N ‐acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N ‐linked glycosylation sites at Asn 43 and Asn 78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn 272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 7766 :301–304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn 272 .