In-Depth Analysis of Human and Mouse Plasma Using 3-D and 4-D Fractionation Strategies

摘要

In-depth profiling of plasma proteomes can potentially identify novel disease biomarkers. But few biomarkers identified by proteomic approaches have advanced to early-stage clinical testing because they often are not sufficiently disease specific. Major challenges in plasma proteome analysis include the very wide dynamic range of protein concentrations, the high protein complexity, and the substantial heterogeneity of most protein concentrations in the normal human population. Because most disease-specific biomarkers are present in blood at very low concentrations, extensive fractionation is required prior to LC-MS/MS analysis. In general, more fractionation will result in greater depth of analysis, but there is a point of diminishing return for each fractionation method and throughput decreases as the number of LC-MS/MS runs per proteome increases. A common feature of most current plasma profiling methods is to first immunodeplete as many high abundance plasma proteins as possible, followed by extensive protein fractionation of the depleted plasma prior to trypsin digestion and LC-MS/MS. In addition, reliable quantitative comparisons are needed for most types of studies. While all quantitative methods have strengths and weaknesses, label-free quantitative comparison of LC-MS signals is increasing in popularity and seems adequately reproducible for most studies. Our laboratory commonly uses two alternative plasma proteome analysis strategies. One powerful approach utilizes a 3-D protein/peptide profiling method consisting of depleting 20 abundant proteins followed by 1-D SDS PAGE, fractionation of the gel lane into 20 to 60 fractions and LC-MS/MS analysis. Proteins can be quantitatively compared using label-free analysis of ion current patterns from the MS full scans. An even greater depth of analysis can be achieved using a 4-D protein/peptide profiling strategy utilizing microscale solution isoelectrofocusing of proteins prior the SDS gel in the 3-D scheme, although throughput is substantially reduced.