DOI: 10.25345/c59k46499
title: MassIVE MSV000095541 - Proteomic analysis of bone marrow supernatants.
title: Sample preparation for proteomics analysis. Bone marrow supernatants (0.5 mL per sample) were concentrated with 3 kDa MWCO Amicon Ultra Centrifugal filter devices (Merck Millipore) up to a final volume of 30 uL. Protease inhibitors were added to the samples and the protein concentration was defined with Bradford Assay. Concentrated samples were processed with the filter aided sample preparation (FASP) method with minor modifications. Briefly, sample volume corresponding to 200 ug of total protein content was mixed with lysis buffer (0.1 M Tris HCl pH 7.6, supplemented with 4% SDS and 0.1 M DTE) and buffer exchange was performed in Amicon Ultra Centrifugal filter devices (0.5 mL, 30 kDa MWCO; Merck Millipore) at 14,000 rcf for 15 min at RT. Each sample was diluted with urea buffer (8 M urea in 0.1 M Tris HCl pH 8.5) and centrifuged.The concentrate was diluted again with urea buffer and centrifugation was repeated. Alkylation of proteins was performed with 0.05 M iodoacetamide in urea buffer for 20 min in the dark, RT, followed by a centrifugation at 14,000 rcf for 10 min at RT. Additional series of washes were conducted with urea buffer (2 times) and ammonium bicarbonate buffer (50 mM NH4 HCO3 pH 8.5, 2 times). Tryptic digestion was performed overnight at RT in the dark, using a trypsin to protein ratio of 1:100. Peptides were eluted by centrifugation at 14,000 rcf for 10 min, lyophilized and stored at -80C until further use. LC-MS/MS analysis Samples were resuspended in 200 uL mobile phase A (0.1% Formic acid ). A 5 uL volume was injected into a Dionex Ultimate 3000 RSLS nano flow system (Dionex, Camberly, UK) configured with a Dionex 0.1 x 20 mm, 5 um, 100 A C18 nano trap column with a flow rate of 5 uL / min. The analytical column was an Acclaim PepMap C18 nano column 75 um x 50 cm, 2 um 100 A with a flow rate of 300 nL / min. The trap and analytical column were maintained at 35C. Mobile phase B was 100% Acetonitrile:0.1% Formic acid. The column was washed and re- equilibrated prior to each sample injection. The eluent was ionized using a Proxeon nano spray ESI source operating in positive ion mode. For mass spectrometry analysis, a Q Exactive Orbitrap (Thermo Finnigan, Bremen, Germany) was operated in MS/MS mode. The peptides were eluted under a 120 min gradient from 2% (B) to 80% (B). Gaseous phase transition of the separated peptides was achieved with positive ion electrospray ionization applying a voltage of 2.5 kV. For every MS survey scan, the top 10 most abundant multiply charged precursor ions between m/z ratio 300 and 2200 and intensity threshold 500 counts were selected with FT mass resolution of 70,000 and subjected to HCD fragmentation. Tandem mass spectra were acquired with FT resolution of 35,000. Normalized collision energy was set to 33 and already targeted precursors were dynamically excluded for further isolation and activation for 15 sec with 5 ppm mass tolerance. MS data processing Raw files were analyzed with Proteome Discoverer 1.4 software package (Thermo Finnigan), using the Sequest search engine and the Uniprot mouse (Mus musculus) reviewed database, downloaded on November 22, 2017, including 16,935 entries. The search was performed using carbamidomethylation of cysteine as static and oxidation of methionine as dynamic modifications. Two missed cleavage sites, a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.05 Da were allowed. False discovery rate (FDR) validation was based on q value: target FDR (strict): 0.01, target FDR (relaxed): 0.05. Normalized serum protein concentrations were imported into R for statistical and quantification analysis. Proteins were considered differentially abundant at a cutoff of |FC| &gt; 1.5, and significant at P &lt; 0.05, as determined by unpaired two-tailed Students t-test. aPDL1 raw files correspond to the treated animals. PBS raw files correspond to the respective control animals.