Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple po
ssible functions that are likely influenced by glycosylation. Identifi
cation of oligosaccharide attachment sites and structural characteriza
tion of oligosaccharides in human serum clusterin has been performed b
y mass spectrometry and Edman degradation. Matrix-assisted laser desor
ption ionization mass spectrometry revealed two molecular weight speci
es of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrom
etry also revealed molecular heterogeneity associated with both the al
pha and beta subunits of clusterin, consistent with the presence of mu
ltiple glycoforms. The data indicate that clusterin contains 17-27% ca
rbohydrate by weight, the alpha subunit contains 0-30% carbohydrate an
d the beta subunit contains 27-30% carbohydrate. Liquid chromatography
electrospray mass spectrometry with stepped collision energy scanning
was used to selectively identify and preparatively fractionate trypti
c glycopeptides. Edman sequence analysis was then used to confirm the
identities of the glycopeptides and to define the attachment sites wit
hin each peptide. A total of six N-linked glycosylation sites were ide
ntified, three in the alpha subunit (alpha(64)N, alpha(81)N, alpha(123
)N) and three in the beta subunit (beta(64)N, beta(127)N, and beta(147
)N). Seven different possible types of oligosaccharide structures were
identified by mass including: a monosialobiantennary structure, bisia
lobiantennary structures without or with one fucose, trisialotriantenn
ary structures without or with one fucose, and possibly a trisialotria
ntennary structure with two fucose and/or a tetrasialotriantennary str
ucture. Site beta(64)N exhibited the least glycosylation diversity, wi
th two detected types of oligosaccharides, and site beta(147)N exhibit
ed the greatest diversity, with five or six detected types of oligosac
charides. Overall, the most abundant glycoforms detected were bisialob
iantennary without fucose and the least abundant were monosialobianten
nary, trisialotriantennary with two fucose and/or tetrasialotriantenna
ry. Clusterin peptides accounting for 99% of the primary structure wer
e identified from analysis of the isolated alpha and beta subunits, in
cluding all Ser-and Thr-containing peptides. No evidence was found for
the presence of O-linked or sulfated oligosaccharides. The results pr
ovide a molecular basis for developing a better understanding of clust
erin structure-function relationships and the role clusterin glycosyla
tion plays in physiological function.