Modified MuDPIT separation identified 4488 proteins in a system-wide analysis of quiescence in yeast

Abstract

A modified multidimensional protein identification technology (MudPIT) separation was coupled to an LTQ Orbitrap Velos mass spectrometer and used to rapidly identify the near-complete yeast proteome from a whole cell tryptic digest. This modified online two-dimensional liquid chromatography separation consists of 39 strong cation exchange steps followed by a short 18.5 min reversed-phase (RP) gradient. A total of 4269 protein identifications were made from 4189 distinguishable protein families from yeast during log phase growth. The "Micro" MudPIT separation performed as well as a standard MudPIT separation in 40% less gradient time. The majority of the yeast proteome can now be routinely covered in less than a days' time with high reproducibility and sensitivity. The newly devised separation method was used to detect changes in protein expression during cellular quiescence in yeast. An enrichment in the GO annotations "oxidation reduction", "catabolic processing" and "cellular response to oxidative stress" was seen in the quiescent cellular fraction, consistent with their long-lived stress resistant phenotypes. Heterogeneity was observed in the stationary phase fraction with a less dense cell population showing reductions in KEGG pathway categories of "Ribosome" and "Proteasome", further defining the complex nature of yeast populations present during stationary phase growth. In total, 4488 distinguishable protein families were identified in all cellular conditions tested.

Figure 1

Proteome coverage of a standard MudPIT and the Micro MudPIT. A. Number of proteins identified in individual runs and the total number identified in three triplicate runs. B. Number of peptides identified in individual runs and the total number identified in three triplicate runs.

Figure 2

Comparison of protein coverage of replicate runs using of standard MudPIT and Micro MudPIT separations. Numbers are the identified proteins in each analysis.

Figure 3

Comparison of the peptide coverage of standard MudPIT separations and Micro MudPIT separations in triplicate runs. Numbers are the identified peptides in each analysis.

Figure 4

A. The median sequence coverage for MudPIT runs is around 19% with a triplicate combined coverage of 25%. The Micro MudPIT coverage is approximately 17% with a triplicate combined coverage of 23%. B. The dynamic range of the Micro MudPIT accessible proteome was estimated using the Exponentially Modified Protein Abundance Index (emPAI) and by using the TAP-fusion library copy numbers. emPAI values and cell copy numbers for proteins detected with 5 spectral counts or above from the combined triplicate Micro MudPIT experiment are displayed in a log-log plot.

Figure 5

A. Proteins identified in individual runs and the total number identified in three triplicate runs for log phase, stationary phase non-quiescent and quiescent cells. B. Same as A, but at the peptide level. C. Median protein sequence coverage of individual and triplicate runs.