151125_Quantitative human cell culture

Protein extracts were reduced with 10ul of 200mM TCEP then alkylated with 10ul or 375mM iodoacetamide in the dark for 30 minutes and TCA/Acetone precipitated (100 ug). Protein pellets were resolubilized in 100ul of 100mM triethyl ammonium bicarbonate (TEAB) and were digested overnight at 37C by adding 10 ug of Trypsin/LysC mixture (V5071, Promega) in 100 mM TEAB. Individual samples (100 ug) were labeled with a unique isobaric mass tag reagent (TMT 10-plex, Thermo Scientific) according to the manufacturer instructions. Both pairing and labeling order of TMT reagent and peptide sample were randomized. Briefly, TMT-6 plex reagents (0.8ug vials) were allowed to come to room temperature before adding 41 ul of anhydrous acetonitrile, then briefly vortexed and centrifuged. The entire TMT reagent vial was added to the 100 ug peptide sample and reacted at room temperature for 1 hr. 5% hydroxylamine (8 ul) was then added to quench the reaction. All TMT labeled samples were combined and vacuum centrifuged to dryness. The combined samples of TMT labeled peptides was resuspended in 2ml of 10mM TEAB and separated into 84 fractions at 250ul/min using a 0- 90% acetonitrile gradient in 10 mM TEAB on a 150mm x 2.1mm ID Waters XBridge 5um C18 using an Agilent 1200 capillary HPLC in normal flow mode and Agilent 1260 micro-fraction collector. The 84 fractions are concatenated into 24 fractions by combining all odd rows of each column 1 through 12 into 12 fractions and all even rows of each column into another 12 fractions. Peptide fractions were resuspended in 20ul 2% acetonitrile in 0.1% formic acid and analyzed by reverse phase liquid chromatography coupled to tandem mass spectrometry. Peptides were separated on a 75 um x 150 mm ProntoSIL-120-5-C18 H column (3µm, 120Å (BISCHOFF), www.bischoff-chrom.com) using 2-90% acetonitrile gradient at 300 nl/min over 90 min on a EasyLC nanoLC 1000 (Thermo Scentific). Eluting peptides were sprayed through 1 µm emitter tip (New Objective, www.newobjective.com) at 2.0 kV directly into a Q-Exactive Plus (QE Plus, Thermo Scientific) mass spectrometer. Survey scans (full ms) were acquired from 350-1700 m/z with data dependent monitoring of up to 15 peptide masses (precursor ions), each individually isolated in a 1.2 Da window and fragmented using HCD activation collision energy 32 and 30 s dynamic exclusion. Precursor and the fragment ions were analyzed at resolutions 70,000 and 35,000, respectively, with automatic gain control (AGC) target values at 3e6 with 50 ms maximum injection time (IT) and 1e5 with 200 ms maximum IT, respectively. Isotopically resolved masses in precursor (MS) and fragmentation (MS/MS) spectra were processor in Proteome Discoverer (PD) software (v1.4, Thermo Scientific). All data were searched using Mascot (2.5.1; www.matrixscience.com) against the Refseq mouse 2012 database. The following criteria were set for all database searches: sample’s species; trypsin as the enzyme, allowing one missed cleavage; cysteine carbamidomethylation and N-terminal TMT label as fixed modifications; TMT label on lysine, methionine oxidation, asparagine and glutamine deamidation as variable modifications. Peptide identifications from Mascot searches were filtered at 1% False Discovery Rate (FDR) confidence threshold, based on a concatenated decoy database search, using the Proteome Discoverer. Proteome Discoverer uses only the peptide identifications with the highest Mascot score for the same peptide matched spectrum from the different extraction methods.