Microscale Phosphoproteome Analysis of 10 000 Cells from Human Cancer Cell Lines

摘要

We developed a miniaturized LC-MS system with a high-recovery phosphopeptide enrichment protocol that allows phosphoproteome analysis of 10~(4) cells. In the enrichment protocol, the key step is to add sodium deoxycholate and sodium lauroyl sarcosinate to the buffer solution for protein extraction and digestion and to omit any subsequent desalt/desurfactant step before phosphopeptide enrichment. The phosphopeptides enriched by hydroxy acid-modified metal oxide chromatography (HAMMOC) are directly injected onto a miniaturized LC column using a nitrogen-pressure-driven cell, instead of switching valve-type injectors. The miniaturized analytical column of 25 (mu)m diameter provided a 3.6-fold improvement in sensitivity over the conventional 100 (mu)m diameter column. Overall, our analytical system provided approximately 80-fold improvement on average in the LC-MS response, and we identified 1011 unique phosphorylated sites based on 995 unique phosphopeptides from a single analysis of 10~(4) HeLa cells (approximately 1 (mu)g of proteins). This is the most sensitive phosphoproteomics system that has so far been reported for proteome-wide analysis of in vivo phosphorylation in mammalian cells.