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

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 mon-itoring 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