DOI: 10.25345/c5w79d
title: MassIVE MSV000086790 - MudPIT LiPMS analyses of whole cell protein extracts from Saccharomyces cerevisiae grown at 21C and 35C
title: Sample Preparation. Three biological replicates of wild type yeast cells (BY4741) were cultured with YEPD either at 21C or 35C overnight (ca. 18hrs) to OD600 around 2. Pre-warmed/chilled fresh YEPD was next used to dilute the cultures, which were grown for an additional 6hrs to OD600 around 1. 120mL of cell culture for each replicate were briefly centrifuged at 3,000 g to collect the cells. Cells were washed once with cold lysis buffer (50mM HEPES, pH 7.5, 150 mM NaCl, 1mM MgCl2, 1% Triton X100) and resuspended in 1mL lysis buffer. The cell suspensions were flash frozen in droplet format in liquid nitrogen and grinded for 3min at 30 Hz. After melting the samples, cell lysates were transferred to 2mL centrifuge tubes and mixed with an additional 800ul of lysis buffer that was used to rinse the grinding jar. Cell lysates were clarified by a 10min centrifugation at 16,000 g at 4C. The concentration of clarified lysate was determined with BCA assay and diluted to 2 mg/mL with buffer (20mM HEPES pH 7.5, NaCl 150mM, 10mM MgCl2). Protein Digestion. 0.9mL of these diluted lysates were digested with proteinase K at 1:100 (w/w) ratio for 5 min at RT. Another 0.9 mL was saved as control without proteinase K digestion. The proteinase K-digested and control samples were immediately terminated by 7 M guanidine hydrochloride and boiled for 5 min. After cooling down to room temperature, samples were reduced with 5 mM TCEP (tris(2-carboxyethyl)phosphine) and alkylated with 10 mM chloroacetamide in dark for 30 min. Samples were diluted with 0.1 M NH4HCO3 to reduce the concentration of GdnHCl to 0.5 M and digested with trypsin (Promega, 18ug, which is 1:50 w/w) for 2hr at 32C with 1mM CaCl2. Additional trypsin (12ug, which is 1:75 w/w) was added to each sample to digest overnight. After overnight digestion, the samples were acidified with formic acid (5%) and were concentrated using Empore Solid Phase Extraction Cartridges prewashed with 1mL of methanol and 1mL of 0.1% Trifluoroacetic acid (TFA). The peptides were eluted with 80% acetonitrile containing 0.1% TFA after washing the cartridge with 1mL of 0.1% TFA. MS Data Acquisition. All peptide samples were dried using a SpeedVac vacuum concentrator and resuspended in 150 ml 5% acetonitrile, 0.1% formic acid (Buffer A). Peptides were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with an Agilent 1260 series quaternary HPLC pump and Velos Pro Orbitrap or Velos Orbitrap Elite mass spectrometers. A fully automated 12-step MudPIT chromatography run was carried out. Each full MS scan (400-1600 m/z) was followed by 15 data-dependent MS/MS scans. MS1 scans were acquired in Orbitrap at 60,000 resolution, while ddMS2 were acquired in the ion trap with Rapid Scan settings. The number of the micro scans was set to 1 for both MS and MS/MS. MS1 AGC target was set to 1.00E+06, MS2 AGC targets at 1.00E+04, and MS2 max injection times at 150 ms. MS1 charge states were between 2-5. MS2 collision energy was set at 35%. Dynamic exclusion settings were: 2 repeat counts; 30 s repeat duration; exclusion list size of 500; and 90 s exclusion duration. MS Data Searching and Processing. Peak files were extracted using RawDistiller and searched using ProLuCID (v. 1.3.3) against a database consisting of 5,846 S. cerevisiae non-redundant proteins (downloaded from NCBI 11-01-2016), 193 usual contaminants (such as human keratins, IgGs, and proteolytic enzymes), and, to estimate false discovery rates (FDRs), 6039 randomized amino acid sequences derived from each non-redundant protein entry. All cysteines were considered as fully carboxamidomethylated (+57.0215 Da statically added), while methionine oxidation was searched as a differential modification. Mass tolerances of 7 ppm for precursor and 800 ppm for fragment ions were used. MS/MS datasets generated for trypsin-digested peptides were searched with KR peptide-end requirements, while no enzyme specificity was required for the MS/MS datasets generated for the proteinase K-digested peptides. DTASelect v1.9 and swallow, an in-house developed software, were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here, all controlled FDRs were less than 1%.