High throughput quantitative analysis of serum proteins using glycopeptide capture and liquid chromatography mass spectrometry.
It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.
Zhang
Hui
H
Institute for Systems Biology, Seattle, WA 98103, USA.
Yi
Eugene C
EC
Li
Xiao-jun
XJ
Mallick
Parag
P
Kelly-Spratt
Karen S
KS
Masselon
Christophe D
CD
Camp
David G
DG
Smith
Richard D
RD
Kemp
Christopher J
CJ
Aebersold
Ruedi
R
D000818:Animals; D001331:Automation; D015415:Biomarkers; D001798:Blood Proteins / Q000737:chemistry*; D002273:Carcinogens / Q000378:metabolism; D002853:Chromatography, Liquid; D016000:Cluster Analysis; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D007477:Ions; D013058:Mass Spectrometry; D051379:Mice; D009374:Neoplasms, Experimental / Q000097:blood* / Q000139:chemically induced / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011336:Probability; D040901:Proteomics / Q000379:methods; D012878:Skin Neoplasms / Q000139:chemically induced; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D015415:Biomarkers; D001798:Blood Proteins; D002273:Carcinogens; D006020:Glycopeptides; D007477:Ions; D010455:Peptides