P69-T Global Analysis of Phosphotyrosine Sites in Src-Transformed Cells: Improved Methods for Sample Preparation and Analysis

Interest in tyrosine phosphorylation patterns has been sparked since increases in phosphotyrosine have been associated with malignancy and tumor formation. We have employed several methods to investigate the phosphotyrosine profile of Src-transformed cells, including an extensive study using the methods published by Rush et al. 1 In order to streamline the Rush et al. protocol, we have generated peptides by running the lysate approximately 1.5 cm into a 10% bis-tris polyacrylamide gel (with reduction and alkylation prior to the gel) followed by in-gel digestion. Phosphotyrosine-containing peptides from tryptic digestions were enriched using either the pY100 antibody or the 4G10 antibody. Phosphotyrosine-containing peptides were eluted off the beads as described by the Rush et al. protocol or with the addition of an acetonitrile elution of the beads. Samples were analyzed on a Thermo LTQ by reverse-phase chromatography using data-dependent analysis. The results were searched using the Sequest algorithm and the mouse subset of the Uniref100 database with concatenated reverse database searching for false-positive estimation. Results were filtered in a two-step process based on multiple Sequest scores, with increased stringency for novel sites. Results from multiple biological replicates were compared with those that we obtained using the Rush et al. protocol, and they produced comparable results. Peptides accepted from multiple replicates resulted in a false-positive rate of less than 3%. The addition of an acetonitrile wash increased the elution of peptides from the beads and increased identifications. This procedure saved 1–2 d for sample preparation with equivalent or improved results. We are currently exploring digestion of whole-cell lysates in trifluoroethanol to further streamline this protocol, and the use of the LTQ-Orbitrap to more confidently identify the sites.