Development of a Label-Free Quantitative Phosphoproteomics Platform Applicable to Non-Cell Culture Biological Matrices

摘要

The most commonly employed LC-MS based quantitative phosphoproteomics experiments are performed using cultured cell lines with incorporated stable-isotope labeled amino acids. Although these systems simplify experimental manipulation and provide relatively large amounts of soluble protein, they do not account for the complexity of signal regulation from other factors or cell types present in the cell's native physiological environment. Here we describe the general analytical and informatic methodologies used for the global quantitation of specific sites of phosphorylation from clinically relevant matrices such as human tissue or biological fluids compatible from a variety of different experimental designs. As an exemplar application of the methodology, a quantitative analysis of lipopolysaccharide challenged and ethyl nitrite protected mouse lung tissue was performed. Phosphopeptide enrichments from 600 ug aliquots of trypsin digested homogenized mouse lung tissue from four unique treatment groups was accomplished using an optimized TiO2 spin-column protocol and were subjected to triplicate 1D-LC-MS/MS analysis on a Waters NanoAcquity UPLC coupled to a Thermo LTQ Orbitrap XL. Following accurate mass and retention time alignment across all LC-MS runs performed within Rosetta Elucidator, a robust mean normalization of precursor ion intensities was applied and area under the curve quantitative measurements were made for all precursor ions. Qualitative identifications were assigned to each precursor ion following Mascot searches against a mus musculus database appended with reverse entries and scoring thresholds were adjusted to yield a peptide 1% FDR. Across all four treatment groups, a total of 778 phosphopeptides were qualitatively identified. Analytical plus TiO2 enrichment variation of pre-digested bovine alpha-casein spiked into each sample prior to enrichment yielded a phosphopeptide intensity variation of.