In-Depth Analysis of a Plasma or Serum Proteome Using a 4D Protein Profiling Method
Comprehensive proteomic analysis of human plasma or serum has been a major strategy used to identify biomarkers that serve as indicators of disease. However, such in-depth proteomic analyses are challenging due to the complexity and extremely large dynamic range of protein concentrations in plasma. Therefore, reduction in sample complexity through multidimensional pre-fractionation strategies is critical, particularly for the detection of low-abundance proteins that have the potential to be the most specific disease biomarkers. We describe here a 4D protein profiling method that we developed for comprehensive proteomic analyses of both plasma and serum. Our method consists of abundant protein depletion coupled with separation strategies – microscale solution isoelectrofocusing and 1D SDS-PAGE – followed by reversed-phase separation of tryptic peptides prior to LC–MS/MS. Using this profiling strategy, we routinely identify a large number of proteins over nine orders of magnitude, including a substantial number of proteins at the low ng/mL or lower levels from approximately 300 μL of plasma sample.
Key wordsPlasma proteome Low-abundance protein Immunoaffinity depletion Biomarker MicroSol IEF SDS-PAGE LC–MS/MS
This work was supported in part by National Institutes of Health Grants CA120393 and CA131582, and institutional grants to the Wistar Institute including an NCI Cancer Core Grant (CA10815) and the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.
- 9.Beer, L. A., Tang, H. Y., Barnhart, K. T., and Speicher, D. W. (2011) Plasma biomarker discovery using 3-D protein profiling coupled with label-free quantitation, Ed. Simpson RJ, Greening DW, Serum/Plasma Proteomics, Methods Mol Biol 728, Humana Press.Google Scholar
- 10.Omenn, G. S., States, D. J., Adamski, M. et al. (2005) Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database, Proteomics 5, 3226–3245.PubMedCrossRefGoogle Scholar