Journal
ANALYTICAL CHEMISTRY
Volume 83, Issue 20, Pages 7698-7703Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac201093g
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Funding
- Yamagata Prefecture
- Tsuruoka City
- Japan Society for the Promotion of Science [23710218]
- Grants-in-Aid for Scientific Research [23710218, 23134507, 21310129] Funding Source: KAKEN
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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.
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