Quantitative Phosphoproteome Analysis of a Macrophage Cell Line Reveals LPS-triggered Cell Signaling Events

摘要

By chelating Fe~(3+) onto Qiagen NTA beads (Ni-NTA spin column, Qiagen), we found these beads to be very effective as an IMAC material for the enrichment of phosphopeptides. In most experiments with this cell line, direct RPLC-ESI-MS analysis of between 500-1000 peptides, more than 90percent of which are identified as phosphopeptides. After separating IMAC enriched phosphopeptide samples by HILIC, and analyzing each fraction using RPLC-ESI-MS, we can identify approximately 4000-5000 unique phosphopeptides from 10 mg of original protein sample. When analyzing SILAC-labeled phosphopeptides, due to the increased complexity of the sample ("Heavy" + "Light"), the number of unique phosphopeptides obtained from each LC-MS run is less than when compared to an unlabeled sample from a single set of conditions. Our preliminary results from a 10 mg protein mixture of LPS-stimulated and unstimulated Raw 264.7 cells have identified 3306 unique phosphopeptides (95percent confidence). The number of unique phosphoproteins was 1387 (search database: Swiss-Prot 56.9). After gene ontology analysis, we found many proteins were known to have functions in innate immunity. For example Thr-203 of ERK-1 (accession number: Q63844) up-regulated 2.4 fold. Ser-915 of MAPKKK1 (accession number: P53349) up-regulated 1.4 fold, Ser-25 of Presenilin-2 (accession number: Q61144) up-regulated 1.5 fold. We also identified candidate proteins that were previously unknown in terms of host cell responses to bacterial pathogenic factors.