Tryptic peptides were digestedfrom HDL fractions isolated by ultracentrifugation
After high-performanceliquid chromatography (HPLC) separation via reversed phase C18 column,the peptides were analyzed by tandem MS. Following a database search,17 proteins were identified shown in SupplementalTable S1, Supporting Information from one replicate as an example
The base −10 log of the expectation that any particular proteinassignment was made at random was lower than −2
ApoA-I wasobserved as the major protein
In humans, about 60% of the proteincontent in HDL is represented by ApoA-I
Other apoproteins were present, including Apo C-II, C-III, D, E,and M, which are typical minor apoproteins associated with HDL
Onthe other hand, ApoB-100, the major constituent of VLDL and LDL, wasnot present
Other proteins known to be associated with HDL were alsoobserved, including acute phase response proteins such as SAA , liversecreted proteins such as alpha-2-HS-glycoprotein ( fetuin A ), andthe key mediators of the complement system C3 inhibitor
The proteins associated with HDL isolated from healthy human plasmain this study have all been suggested as HDL-associated proteins inprevious studies
These results confirmed our HDL isolation approach and provideda list of proteins for site-specific glycosylation analysis