Project PXD000293

PRIDE Assigned Tags:
Biological Dataset
Dataset Belongs to:
PRIME-XS Project

Summary

Title

Ti4+-IMAC label-free quantification

Description

We combine high-resolution mass spectrometry with Ti4+-IMAC phosphopeptide enrichment and label-free quantification to monitor the phosphoproteome of Jurkat T-cells following stimulation by Prostaglandin E2. Jurkat T lymphoma cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (Lonza). For PGE2 stimulation, cells were centrifuged for 1 min at 1500g. The growth medium was removed and the cells were resuspended at a final concentration of 1-2 × 106 cells/ml in RMPI and supplemented with 0 (control) or 10 uM PGE2 and incubated for 5, 10, 20, 30 or 60 min. After cell lysis, reduction and alkylation, proteins were digested with Lys-C and Trypsin. The digests were desalted using Sep-Pak C18 cartridges, dried in vacuo and stored at −80 °C for further use. Phosphopeptide enrichment was performed as described in Zhou H, Ye M, Dong J, Corradini E, Cristobal A, Heck AJ, Zou H, Mohammed S. Nat Protoc. 2013 Mar;8(3):461-80. The enriched phosphopeptides were subjected to a reversed phase nano-LC–MS/MS analysis consisting of a Proxeon EASY-nLC 1000, an analytical column heater (40°C) and an LTQ-Orbitrap Elite. After the survey scans, the 20 most intense precursors were selected for subsequent CID or ETD-IT fragmentation. A programmed data-dependent decision tree determined the choice of the most appropriate technique for a selected precursor. In essence, doubly charged peptides were subjected to CID fragmentation and more highly charged peptides were fragmented using ETD. Raw data were processed with MaxQuant version 1.3.0.523, and the peptides were identified from the MS and MS/MS spectra searched against a concatenated forward-decoy Swissprot Homo sapiens database version 2012_09 (40,992 sequences) using the Andromeda search engine. The database search was performed with the following parameters: an initial mass tolerance of ±20 ppm for precursor masses; final mass tolerance of ±6 ppm: ±0.6 Da for CID and ETD-ion trap fragment ions, allowing two missed cleavages. Cysteine carbamidomethylation was used as a fixed modification and methionine oxidation, protein N-terminal acetylation and serine, threonine and tyrosine phosphorylation as variable modifications. For the identification, the false discovery rate was set to 0.01 for peptides, proteins and sites and the minimum peptide length allowed was six amino acids and a minimum peptide score of 60. The match between run feature was set on. A site localization probability of at least 0.75 and a score difference of at least 5 were used as threshold for the phosphoresidue localization. Normalization was performed by subtracting the median of log transformed intensities for each LC-MS/MS run.

Sample Processing Protocol

HeLa cells were grown to confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (Lonza), hereafter cells were washed twice with PBS and harvested. Jurkat T lymphoma cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (Lonza). Before PGE2 stimulation, cells were centrifuged for 1 min at 1500g, growth medium was removed and the cells were resuspended at a final concentration of 1-2 × 106 cells/ml in RMPI. Next, cells were supplemented with 0 (control) or 10 μM PGE2 and incubated for 5, 10, 20, 30 or 60 min. After treatment Jurkat cells were washed twice with PBS and harvested. Cell lysis was performed on ice by sonication in buffer containing 50 mM ammonium bicarbonate (pH 8.0), 8 M urea, 1 mM sodium orthovanadate, complete EDTA-free protease inhibitor mixture (Roche) and phosSTOP phosphatase inhibitor mixture (Roche). Cell debris were then removed by centrifugation at 20 000g for 15 min at 4 °C. The total protein concentration was measured using a Bradford Assay (BioRad) and then split into 1 mg aliquots for enzymatic digestion. Proteins were reduced with DTT at a final concentration of 4 mM at 56 °C for 25 min; subsequently samples were alkylated with iodoacetamide at a final concentration of 8 mM at RT for 30 min in the dark. Proteins were then digested using Lys-C (1 μg Lys-C per 75 μg protein) and incubated for 4 h at 37 °C. The solution was then diluted to a final urea concentration of 2 M with 50 mM ammonium bicarbonate, and trypsin was added (1 μg trypsin per 100 μg protein) and incubated at 37 °C overnight. The digestion was quenched by acidification to 5% formic acid. The digests were desalted using Sep-Pak C18 cartridges, dried in vacuo and stored at −80 °C for further use. The Ti4+-IMAC beads (500 μg of beads/200 μL pipet tip) were loaded onto GELoader tips (Eppendorf) using a C8 plug. To reduce variations of enrichment processes, in parallel spin tip enrichment was used. The Ti4+-IMAC columns were conditioned using 50 μL of loading buffer consisting of 80% acetonitrile (ACN)/6% trifluoroacetic acid (TFA) and centrifugation at 200g for 10 min. The protein digests were dissolved in 80% ACN/6% TFA and split in aliquots corresponding to ~100 μg and ~250 μg for the HeLa and Jurkat cell lysates, respectively. The aliquots were transferred to the spin tips and centrifuged at 100g for 30 min. Then, the columns were sequentially washed with 50 μL of washing buffer 1 (50% ACN, 0.5% TFA containing 200 mM NaCl) and additional washing with 50 μL of buffer 2 of 50% ACN/0.1% TFA, each centrifuged at 170g for 15 min, respectively. The bound peptides were eluted into a new tube (already containing 35 μL of 10% formic acid) with 20 μL of 10% ammonia by centrifugation at 100g for 20 min. A final elution was performed with 5 μL of 80% ACN/2% formic acid for 10 min. The collected eluate was further acidified by adding 3 μL of 100% formic acid prior to nLC-MSMS analysis. Peptides were subjected to reversed phase nLC–MSMS analysis using a Proxeon EASY-nLC 1000 (Thermo Scientific, Odense, Denmark) with an analytical column heater (40°C) and an LTQ-Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany). Peptides were first trapped (Dr Maisch Reprosil C18, 3 μm, 2 cm x 100 μm) at a maximum pressure of 800 bar with 100% solvent A (0.1 % formic acid in water) before being separated on the analytical column (either Agilent Poroshell 120 EC-C18, 2.7 μm, 40 cm x 50 μm for the HeLa cell samples or Agilent Zorbax SB-C18, 1.8 μm, 40 cm x 75 μm for the Jurkat T cell samples). Peptides were chromatographically separated by a 90 min gradient from 7% to 30% solvent B (0.1% formic acid in ACN) at a flowrate of 150 or 100 nL/min. The total measurement time for each sample was 110 min. The eluent was sprayed via a distal coated fused silica emitter (360 µm o.d., 20 µm i.d., 10 µm tip i.d.; constructed in-house) butt-connected to the analytical column. The electrospray voltage was set to 1.7 kV. The mass spectrometer was operated in a data-dependent mode to automatically switch between MS and MS/MS. Briefly, survey full-scan MS spectra were acquired in the Orbitrap analyzer, scanning from m/z 350 to m/z 1500 at a resolution of 60,000 at m/z 400 using an AGC setting of 1e6 ions. Charge state screening was enabled and precursors with either unknown or 1+ charge states were excluded. After the survey scan the 20 most intense precursors were selected for subsequent decision tree-based iontrap CID or ETD fragmentation.(18) The normalized collision energy for CID was set to 35% and supplemental activation for ETD and dynamic exclusion were enabled (exclusion size list 500, exclusion duration 40 s).

Data Processing Protocol

Raw data were processed with MaxQuant version 1.3.0.5, MS and MSMS spectra were searched against a concatenated forward-decoy Swissprot Homo sapiens database version 2012_09 (40,992 sequences) using the Andromeda search engine. The database search was performed with the following parameters: an initial mass tolerance of ±20 ppm and a final mass tolerance of ±6 ppm for precursor masses, ±0.6 Da for CID and ETD ion trap fragment ions, allowing two missed cleavages. Cysteine carbamidomethylation was used as a fixed modification and methionine oxidation, protein N-terminal acetylation and serine, threonine and tyrosine phosphorylation as variable modifications. The false discovery rate was set to 0.01 for peptides, proteins and phosphosites, the minimum peptide length allowed was six amino acids and a minimum Andromeda peptide score of 60 was required. The match between run feature was enabled. A site localization probability of at least 0.75 and a score difference of at least 5 were used as threshold for the localization of phosphoresidues. Normalization was performed by subtracting the median of log transformed intensities for each nLC-MSMS run.

Contact

Piero Giansanti, NPC
Albert J. R. Heck, Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands ( lab head )

Submission Date

12/05/2014

Publication Date

22/05/2014

Corresponding dataset(s) in other omics resources

Dataset visualisation in external resources

PPV000010 (MS-Viewer)

Publication

    de Graaf EL, Giansanti P, Altelaar AF, Heck AJ. Single step enrichment by Ti4+-IMAC and label free quantitation enables in-depth monitoring of phosphorylation dynamics with high reproducibility and temporal resolution. Mol Cell Proteomics. 2014 May 21. pii: mcp.O113.036608 PubMed: 24850871