Deep and Highly Sensitive Proteome Coverage by LC-MS/MS Without Prefractionation

摘要

In-depth MS-based proteomics has necessitated fraction-ation of either proteins or peptides or both, often requiring considerable analysis time. Here we employ long liquid chromatography runs with high resolution coupled to an instrument with fast sequencing speed to investigate how much of the proteome is directly accessible to liquid chromatography-tandem MS characterization without any prefractionation steps. Triplicate single-run analyses identified 2990 yeast proteins, 68% of the total measured in a comprehensive yeast proteome. Among them, we covered the enzymes of the glycolysis and gluconeogen-esis pathway targeted in a recent multiple reaction monitoring study. In a mammalian cell line, we identified 5376 proteins in a triplicate run, including representatives of 173 out of 200 KEGG metabolic and signaling pathways. Remarkably, the majority of proteins could be detected in the samples at sub-femtomole amounts and many in the low attomole range, in agreement with absolute abundance estimation done in previous works (Picotti et al. Cell, 138, 795-806, 2009). Our results imply an unexpectedly large dynamic range of the MS signal and sensitivity for liquid chromatography-tandem MS alone. With further development, single-run analysis has the potential to radically simplify many proteomic studies while maintaining a systems-wide view of the proteome.