Proteomics Analysis

Phosphoproteomics experiments were performed using mass spectrometry as previously reported.13 In brief, cells were lysed in urea lysis buffer (8M urea, 10 mM Na₃VO₄, 50 mM NaF, 100 mM β-glycerol phosphate, and 25 mM Na₂H₂P₂O₇), and proteins reduced and alkylated by sequential addition of 1 mM DTT and 5 mM iodoacetamide. Immobilized trypsin was then added to digest proteins into peptides. After overnight incubation with trypsin, peptides were desalted by solid-phase extraction (SPE) using OASIS HLB columns (Waters) in a vacuum manifold following manufacturer’s guidelines with the exception that the elution buffer contained 1M glycolic acid. Phospho-peptides were enriched from the resulting peptide mixture using TiO₂ chromatography with the modifications described by Montoya.14 TiO₂ chromatographic media were added to the SPE eluted peptides and incubated 5 min with rotation. The TiO₂ media were then packed in empty spin-tips and washed three times with 1M glycolic acid, 5% trifluoroacetic acid (TFA). Phospho-peptides were eluted with 5% NH₄OH and dried in a vacuum concentrator.

Dried phospho-peptide extracts were dissolved in 0.1% TFA and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an LTQ-orbitrap as described before.13 Gradient elution was from 2% to 35% buffer B in 90 min with buffer A (0.1% formic acid in water and buffer B was 0.1% formic acid in acetonitrile) being used to balance the mobile phase. MS/MS was acquired in multistage acquisition mode. MS raw files were converted into Mascot Generic Format using Mascot Distiller (version 1.2) and searched against the SwissProt database (Database: 2013.03) restricted to human entries using the Mascot search engine (version 2.3). Allowed mass windows were 10 ppm and 600 mmu (millimass units) for parent and fragment mass-to-charge values, respectively. Variable modifications included in searches were oxidation of methionine, pyro-glu (N-term), and phosphorylation of serine, threonine, and tyrosine. Results were filtered to include those with a potential for false discovery rate less than 1% by comparing with searches against decoy databases. Quantification was performed by obtaining peak areas of extracted ion chromatographs (XICs) for the first three isotopes of each peptide ion using Pescal.15 Mass and retention time windows of XICs were 7 ppm and 1.5 min, respectively.