Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry.
Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3-5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.
Adkins
Joshua N
JN
Biological Sciences Department, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Varnum
Susan M
SM
Auberry
Kenneth J
KJ
Moore
Ronald J
RJ
Angell
Nicolas H
NH
Smith
Richard D
RD
Springer
David L
DL
Pounds
Joel G
JG
D001798:Blood Proteins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D019295:Computational Biology; D019075:Electrophoresis, Capillary; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D010449:Peptide Mapping; D020543:Proteome; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism
D001798:Blood Proteins; D020543:Proteome; D014357:Trypsin