DOI: 10.25345/c5vh5cx7c
title: MassIVE MSV000100454 - The Proteome Landscape of Replicative Aging: Spatiotemporal Dynamics Across Cellular Compartments
title: Sample Preparation: Three biological replicates of wild-type yeast cells (BY4741) were cultured with YEPD at 30C overnight in glass tubes on rotating drums to OD600 around 0.6. About 3 OD600 were collected by centrifugation (3000g, 20sec) as M-cells for biotinylation labeling of their cell wall. Biotin solution was added to the cells to 1mg/ml, gently rock for 30min at RT. Then resuspend the cells in PBS pH7.2, added 3050 l BioMag beads.Culture cells and change medium every 6-8hr. Harvest the aged M-cells by magnet bar. Young cells were prepared as following: By4741 cells were cultured in YPD overnight. Refresh cell to OD 600 of ~0.2, and culture for 2-3hr until OD 600 of ~0.6. Collect cells and add BioMag beads. Pre-warmed/chilled fresh YEPD was used to dilute the cultures, which were grown for an additional 6hrs to OD600 around 1. Cells were washed once with cold lysis and resuspended in 1mL lysis buffer. The cell suspensions were flash frozen as droplets in liquid nitrogen. Cell lysates were mixed with 800uL of lysis buffer. Cell lysates were clarified by a 10min centrifugation at 16000g. The concentration of clarified lysate was determined with BCA assay and diluted to 2 mg/mL. Protein Digestion: Cells were washed with cold lysis and resuspended in lysis buffer. The cell suspensions were flash frozen as droplets in 50mL grinding jars in liquid nitrogen and grinded for 10 cycles. The concentration of lysate was determined with BCA assay and were digested with 8.5ug Proteinase K for 5min at room temperature. The digestion was quenched by 7M GdnHCl and boiled for 5 min. Samples were reduced with 5mM TCEP and alkylated in dark for 30 min. Add 0.1 M NH4HCO3 to reduce the concentration of GdnHCl to 0.5 M and digested with trypsin for 2hr at 37C with 1mM CaCl2. Additional trypsin was added to each sample to digest overnight. The samples were clean up by Empore Solid Phase Extraction Cartridges. Peptides were dried using a SpeedVac and resuspended in water for peptide quantification. TMT labeling and MS Data Acquisition: Each TMT6plex vial was reconstituted in 40 ul anhydrous acetonitrile. 18ug of peptides from each sample were mixed with 20uL of the appropriate TMT reagent. Labeling reactions were incubated for 1hr at room temperature. High pH reverse phase fractionation: The combined TMT labeled peptide mixture was resuspended in 300uL of 0.1% TFA. Peptide solution was loaded onto high pH reverse phase fractionation the cartridge. 9 fractions were sequentially eluted into new collection tubes using 300uL of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 50%, and 80% acetonitrile in 0.1% TFA. All fractions were evaporated to dryness by speedvac and subsequently reconstituted in buffer A prior to LCMS analysis. Mass spectrometry data acquisition: TMT labeled peptide fractions were analyzed on an Orbitrap Eclipse Tribrid Mass Spectrometer (Thermo Fisher Scientific). Peptides were first loaded onto an Acclaim PepMap100 C18 trap cartridge at a flow rate of 8uL/min using the loading pump. Analytical separation was performed using a 75um microcapillary column packed in-house with 250mm of 1.9um ReproSil-Pur C18 AQ resin. Column temperature was maintained at 50C. Mobile phase A consisted of H2O:ACN:FA(95:5:0.1, v/v/v), and mobile phase B consisted of H2O:ACN:FA (20:80:0.1, v/v/v). The nanoLC flow rate was set to 0.18uL/min. The Orbitrap Eclipse was operated in TMT SPS MS3 mode. Full MS scans were acquired in the Orbitrap at 120,000 resolution. MS2 spectra were generated by CID at 35% normalized collision energy and detected in the ion trap using turbo scan mode. Dynamic exclusion was 45 sec. For TMT quantification, SPS was applied by selecting the top 10 MS2 fragment ions, followed by HCD at 65% NCE and detection of MS3 spectra in the Orbitrap at a resolving power of 50,000. Database searching and data analysis: LC MS/MS data were processed using Proteome Discoverer version 3.1. MS/MS spectra were searched against a non-redundant Saccharomyces cerevisiae protein database downloaded from NCBI (January 12, 2022), supplemented with common contaminant proteins. Database searching was performed using SEQUEST with the following parameters: no enzyme specificity, precursor mass tolerance of 10ppm, fragment mass tolerance of 0.3Da, and up to two missed cleavages. Carbamidomethylation of C (+57.021 Da) and TMT labeling of K and peptide N-termi(+229.163 Da) were specified as static modifications, while oxidation of M(+15.995 Da) was set as a dynamic modification. Peptide-spectrum matches were filtered to FDR of 1% at the peptide level using Percolator. Reporter ion intensities were extracted from MS3 spectra, and protein quantification was performed by summing reporter ion intensities across all corresponding PSM.