A method for the identification of glycoproteins from human serum by a combination of lectin affinity chromatography along with anion exchange and Cu-IMAC selection of tryptic peptides.
This paper reports a method for identifying glycoproteins from human serum. Glycoproteins were selected with a concanavalin A (Con A) lectin column and then tryptically digested prior to sequential chromatographic selection of acidic and histidine containing peptides. Acidic peptides were selected with a strong anion exchange (SAX) column. Peptides captured by the SAX columns were then released and histidine-containing peptides in the mixture selected with a copper loaded immobilized metal affinity chromatography (Cu-IMAC) column. This serial chromatographic selection process reduced the complexity of proteolytic digests by more than an order of magnitude. Peptides selected by this serial process were then fractionated by reversed-phase chromatography (RPC) and identified by tandem mass spectrometry. The method was initially validated using human transferrin before application to human serum. The results show that all the peptides identified except one contained histidine and acidic amino acids.
Qiu
Ruiqing
R
Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA. qiur@purdue.edu
Zhang
Xiang
X
Regnier
Fred E
FE
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003208:Concanavalin A; D003300:Copper / Q000737:chemistry; D006023:Glycoproteins / Q000097:blood* / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014168:Transferrin / Q000032:analysis; D014357:Trypsin / Q000378:metabolism
D006023:Glycoproteins; D010446:Peptide Fragments; D014168:Transferrin; D003208:Concanavalin A; D003300:Copper; D014357:Trypsin