Ultra-deep human phosphoproteome reveals different regulatory nature of Tyr and Ser/Thr-based signaling
Regulatory protein phosphorylation controls nearly every normal and pathophysiological signaling system in eukaryotic cells. Despite great advances in mass spectrometry based-proteomics, the total number, localization and site-specific stoichiometry of this post-translational modification (PTM) are unknown. Here we develop stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. Label-free quantitation determined very high stoichiometries in mitosis or growth factor signaling and more than three-quarters of cellular proteins were detected as phosphoproteins. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites only saturate for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is statistically enriched on higher abundance proteins and this correlates with the substrate Km values of tyrosine kinases. Our data suggests that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes.
Sample Processing Protocol
HeLa S3 cells were subjected to a double thymidine block in combination with nocodazole to obtain a homogenous mitotic phase population and the efficiency of mitotic arrest was monitored by FACS analysis. Cells were washed and suspended in PBS and treated with 100ng/ml EGF for 5 or 15 min. For tyrosine inhibition of phosphatases, cells were treated with 1mM pervanadate and 50 ng/ml calyculin A for 15 min at 37°C. A total of 12.5 mg of protein lysate per experiment was digested by the FASP method. For proteome analysis, 30 μg of the peptides were separated on a pipette-tip based SAX column. For phosphopeptide enrichment 6mg peptides obtained from FASP were fractionated by strong cation exchange (SCX) chromatography and subjected to phosphopeptide enrichment using TiO2 beads. Briefly, peptides were dissolved in 80% acetonitrile (ACN) and 6% triflouroacetic acid (TFA) and incubated with TiO2 beads for 20 minutes and washed with 80% ACN and 0.1% TFA before phosphopeptide elution under basic pH using ammonia. For P-Tyr enrichment, 8mg of digested peptides were subjected to TiO2 enrichment before immunoprecipitation with a mix of anti-P-Tyr antibodies as described. The (phospho)peptides were desalted on C18 StageTips. Peptides were separated on a 50 cm reversed phase column (75 μm inner diameter, packed in-house with ReproSil-Pur C18-AQ 1.9μm resin (Dr. Maisch GmbH)) over a 120 or 240-min gradient of 5–60% buffer B (0.1% (v/v) formic acid, 80% (v/v) acetonitrile) using the Proxeon Ultra EASY-nLC system. The LC system was directly coupled on-line with a Q Exactive instrument (Thermo Fisher Scientific) via a nanoelectrospray source. The mass spectrometer was programmed to acquire in a data dependent mode using a fixed ion injection time strategy. Full scans were acquired in the Orbitrap mass analyzer with resolution 70,000 at 200m/z. For the full scans, 3E6 ions were accumulated within a maximum injection time of 20 ms and detected in the Orbitrap analyzer. The ten most intense ions with charge states ≥ 2 were sequentially isolated to a target value of 1e6 with a maximum injection time of 60 ms or 80 ms and fragmented by HCD in the collision cell (normalized collision energy of 25%) and detected in the Orbitrap analyzer at 17,500 resolution.
Data Processing Protocol
Raw mass spectrometric data was analyzed in the MaxQuant environment version 184.108.40.206, and employed Andromeda for database search. The MS/MS spectra were matched against the human Uniprot FASTA database version 2/25/2012 (81213 entries). Enzyme specificity was set to trypsin and the search included cysteine carbamidomethylation as a fixed modification and N-acetylation of protein, oxidation of methionine and/or phosphorylation of Ser, Thr, Tyr residue (STY) as variable modifications. Up to two missed cleavages were allowed for protease digestion and peptides had to be fully tryptic.
Sharma K, D'Souza RC, Tyanova S, Schaab C, Wiśniewski JR, Cox J, Mann M. Ultradeep Human Phosphoproteome Reveals a Distinct Regulatory Nature of Tyr and Ser/Thr-Based Signaling. Cell Rep. 2014 Aug 20. pii: S2211-1247(14)00620-2 PubMed: 25159151