BY4741 wild-type yeast were grown in yeast extract peptone dextrose media to mid-log phase (OD600 = 0.6). Proteins were chemically extracted with YPer (Thermo Scientific Pierce; Rockford, IL), and digested with Promega sequencing-grade modified trypsin (Madison, WI) at a 1:50 enzyme:substrate ratio at 37 °C overnight and quenched by acidification with TFA. Peptides were desalted and labeled with Thermo Scientific Pierce TMTsixplex (Rockford, IL; lot number KD130680A), with intermittent mixing at room temperature, and quenched following an hour of incubation. Peptides labeled with tags of nominal m/z 126 through 131 were mixed in ratios of 1: 5: 2: 1.5: 1: 3, respectively.
Peptides were separated on a Waters nanoACQUITY UPLC (Milford, MA) with a self-packed 9 cm precolumn (75 μm i.d.) and a 30 cm analytical column (50 μm i.d.), both packed with Alltech Alltima 5 μm C18 particles (Deerfield, IL) [33 (link)]. The peptides were eluted with a gradient of 5 to 30% acetonitrile over two hours at a flow rate of 300 nL/min. The eluent was analyzed with LC–MS/MS on a Thermo Scientific LTQ Orbitrap Velos mass spectrometer (San Jose, CA/Bremen, Germany). The instrument method was 165 minutes and consisted of a 30,000 resolving power MS1 survey scan followed by data-dependent top-10 higher-energy collision dissociation (HCD) MS2 at 7,500 resolving power, all detected in the orbitrap. Precursor charges states that were unknown or +1 were excluded, and dynamic exclusion was enabled after one fragmentation event for 45 seconds.
This dataset was searched against the Saccharomyces Genome Database [36 (link)] (http://www.yeastgenome.org/ ; January 5, 2010 release; “all” file including verified, uncharacterized, and dubious open reading frames, and pseudogenes). Full trypsin enzymatic specificity was required, allowing up to three missed cleavages. Carbamidomethylation of cysteines (+57 Da) and TMT 6-plex on peptide N-termini and lysines (+229 Da) were specified as fixed modifications, while oxidation of methionines (+16 Da) and TMT 6-plex (+229 Da) on tyrosines were specified as variable modifications. An average mass tolerance of ±5 Da was used for precursors, while a monoisotopic mass tolerance of ±0.01 Da was used for products. For TPP analysis, the data was searched with Sequest (version 27 from the University of Washington) or OMSSA (2.1.9) using either a ±5 Da average precursor mass tolerance or a ±10 ppm monoisotopic precursor mass tolerance and a monoisotopic fragment bin size of 0.01 Da (Sequest) or a monoisotopic product mass tolerance of ±0.01 Da (OMSSA). Results were filtered with PeptideProphet, iProphet and ProteinProphet from TPP 4.3 rev 1. The accurate mass, non-parametric model, and decoy estimation options were used in PeptideProphet. The TPP component Libra was used for isobaric label quantitation.
Peptides were separated on a Waters nanoACQUITY UPLC (Milford, MA) with a self-packed 9 cm precolumn (75 μm i.d.) and a 30 cm analytical column (50 μm i.d.), both packed with Alltech Alltima 5 μm C18 particles (Deerfield, IL) [33 (link)]. The peptides were eluted with a gradient of 5 to 30% acetonitrile over two hours at a flow rate of 300 nL/min. The eluent was analyzed with LC–MS/MS on a Thermo Scientific LTQ Orbitrap Velos mass spectrometer (San Jose, CA/Bremen, Germany). The instrument method was 165 minutes and consisted of a 30,000 resolving power MS1 survey scan followed by data-dependent top-10 higher-energy collision dissociation (HCD) MS2 at 7,500 resolving power, all detected in the orbitrap. Precursor charges states that were unknown or +1 were excluded, and dynamic exclusion was enabled after one fragmentation event for 45 seconds.
This dataset was searched against the Saccharomyces Genome Database [36 (link)] (
