Modified MuDPIT Separation Identified 4488 Proteins in a System-wide Analysis of Quiescence in Yeast

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.