P195-T Reproducible Label-Free Quantitative Analysis of Low-Abundance Plasma Proteins using 3D Protein/Peptide Analysis Coupled with Protein Expression Analysis Software

Quantitative LC-MS/MS analyses of human plasma are predominantly challenged by the wide dynamic range of proteins and extensive biological sample variation, which make systematic detection and quantitation of low-abundant proteins for biomarker discovery quite difficult. In a proof-of-concept study, plasma samples were processed by alternative multi-dimensional workflows that first utilized immunodepletion of twenty abundant plasma proteins (ProteoPrep 20, Sigma). Multiple recombinant proteins were spiked into depleted plasma samples at varying levels to determine detection limits of subsequent SDS-PAGE + LC-MS/MS vs. LC-MS/MS only. After SDS-PAGE, entire lanes were cut into uniform slices, and each slice was digested with trypsin. Digests were subsequently analyzed by nanocapillary reverse-phase chromatography directly coupled to an ESI-LTQ-FT mass spectrometer (Thermo) operating in data-dependent mode. In addition, aliquots of each sample were either digested in solution or as single in-gel digests after brief electrophoresis in an SDS gel, to evaluate advantages and complications of multi-fraction vs. single-fraction LC-MS/MS analysis of a proteome. Resultant LC-MS/MS data were analyzed in a blinded fashion using the Elucidator protein expression data-analysis system. Using the PeakTeller algorithm and statistical analysis tools, we were able to effectively organize the data to distinguish controls from spiked samples and estimate the relative abundances of spiked-in proteins in different samples. Detection limits, protein coverage, and reproducibility of the alternative workflows were compared. This study demonstrates that reproducible relative quantitation can be achieved in complex mixtures such as plasma, using a combination of abundant protein depletion followed by different workflow approaches and data analysis using the Elucidator system.