PNNL

Abstract

Blood serum is a complex bodily fluid that contains proteins ranging in concentration over at least nine orders of magnitude. Using a combination of powerful mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with sub-mL 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 online 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 an increase in the number of proteins detected in an individual serum sample by 3 to 5 fold. 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 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.