1. Propagate HEK293T cell in RPMI 1640 media supplemented with 2 mM L-glutamine, 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2humidified incubator until five 15-cm cell culture treated plates are grown to 90% confluence.
|Suggestion| Five 15-cm plates of HEK293T provide ~10 mg of protein lysates. Different cell lines may require different number of plates to provide the desired amount of proteome.
2. Remove media and wash cells once with DPBS.
3. Add 3 mL of DPBS to each plate and gently collect cells from plates with sterile scraper.
4. Wash cells two more times with DPBS by centrifugation at 200gfor 2 minutes.
5. Resuspend cells in 4 mL of DPBS with EDTA-free protease inhibitor tablet and 1:100 phosphatase inhibitor cocktail 3.
6. Divide live cells into ten aliquots of 400 mL each (or 40 aliquots of 100 mL each if thermal cycler tubes are used).
In Situ Thermal Pulse
7. Expose each 400 mL of cell lysates to a steady temperature between 37°C to 67°C for 3 mins in parallel (eg. aliquot #1 at 37°C for 3 mins, aliquot #2 at 40°C for 3 mins, etc.)
|Caution| Cells must be exposed to temperature treatment immediately after collection from plates and all aliquots must be treated in parallel to avoid sample variation due to time delay.
8. After heat treatment, cool and incubate cells at 25°C for another 3 mins.
9. Immediately lyse cells by rapid freeze-thawing three times with liquid nitrogen.
10. Remove insoluble proteins and cell debris by centrifugation at 17,000gfor 10 mins.
11. Set aside a small amount of supernatant from the 37°C aliquot for protein quantification by Bradford assay.
|Caution| For high efficiency phosphopeptide enrichment, at least 700 mg of proteins (preferably 1 mg) should be present in the 37°C aliquot.
12. Collect 360 mL of lysate from each aliquot. Be careful not to disturb the pellet.
13. Denature lysates in 8 M urea, followed by disulfide reduction with DTT (10 mM, 30 mins, 65°C), alkylation (iodoacetamide, 15 mM, 30 min, room temperature, protected from light) and quenching (DTT, 5 mM, 10 mins, room temperature).
14. Dilute lysates 4-fold in ammonium bicarbonate solution (50 mM, pH 8.0).
15. Add CaCl2(final concentration 1 mM) and sequencing grade trypsin (~1:100 enzyme/protein ratio) to each aliquot.
16. Rotate at 37°C while rotating overnight.
17. Quench peptide digestion reaction by acidification to pH 2-3 with 1% formic acid.
18. Desalt peptides with Sep-Pak tC18 cartridges according to manufacturer’s protocol.
19. Dry eluted peptides under vacuum and resuspend in 1 mL of LC-MS grade water.
20. Reserve 50 mL or 5% from each aliquot for bulk unmodified proteome thermal profiling.
21. Freeze and lyophilize the two sets of peptides (95% and 5%).
22. Resolubilize the major fraction of lyophilized peptides (95%) in 28.5% lactic acid, 57% acetonitrile, 0.28% TFA, and applied to TiO2spin tips (88303, Thermo Pierce) for phosphopeptide enrichment according to manufacturer’s protocol.
23. Acidify eluted phosphopeptides with 1% formic acid until pH 2-3.
24. Desalt acidified phosphopeptides with ZipTip C18 tips according to manufacturer’s protocol.
25. Freeze and lyophilize desalted phosphopeptides.
|Suggestion| Desalted peptides could be stored -20°C for months but modified peptides are more sensitive to storage and freeze-thaw cycles. For best results, modified peptides should be analyzed right away.
26. Reconstitute TMT reagents to 8 mg/mL with anhydrous acetonitrile.
27. Re-dissolve lyophilized peptides in 100 mL of 200 mM HEPES buffer, pH 8.0. Sonicate with water bath to help with reconstitution.
28. Add 10 mL of reconstituted TMT reagents to each aliquot of peptides. Each aliquot should be labeled by a different isobaric TMT tag.
29. Incubate for 1 hour in room temperature with gentle shaking.
30. Quench reaction by adding 5 mL of 5% hydroxylamine solution to each tube.
31. Incubate for 15 mins in room temperature with gentle shaking.
32. Combine all 10 TMT-labeled phosphopeptides into one tube, and combine all 10 TMT-labeled non-enriched peptides into another tube.
33. Acidify the peptide solution by adding formic acid until pH 2-3.
34. Desalt using ZipTip C18 tips according to manufacturer’s protocol.
35. Freeze and lyophilize.
Proteomic Analysis by LC-MS/MS
36. Dissolve processed peptides in LC-MS/MS Buffer A (LC-MS/MS grade H2O with 0.1% formic acid). Sonicate for 5 mins to help peptides dissolve if needed.
37. Perform LC-MS/MS with an Easy-nLC 1000 ultra-high pressure LC system (ThermoFisher) using a PepMap RSLC C18 column (75 μm x 50 cm; 3 μm, 100 Å, Thermo Scientific) heated to 40°C coupled to a Q Exactive HF orbitrap and Easy-Spray nanosource (ThermoFisher).
38. Separate samples using the following gradient of buffer B (0.1% Formic acid acetonitrile) at 300 nL/min: 0-10% buffer B in 5 minutes, 10-40% buffer B in 240 mins, 40-90% buffer B over 6 mins, and hold at 90% for 20 mins.
39. Collect MS/MS spectra from 0 to 250 mins using a data-dependent, top-10 ion setting with the following settings: acquire full MS scans at a resolution of 120,000, scan range of 375-1500 m/z, maximum IT of 60 ms, AGC target of 1e6, and data collection in profile mode. Perform MS2 by HCD fragmentation with a resolution of 60,000, AGC target of 1e5, maximum IT of 60 ms, NCE of 30, and data type in centroid mode. Set isolation window for precursor ions to 1.0 m/z with an underfill ratio of 0.5%. Exclude peptides with charge state 1 and undefined and set dynamic exclusion to 20 seconds. Set S-lens RF level to 60 with a spray voltage value of 2.60kV and ionization chamber temperature of 300°C.
|Suggestion| Chromatography and MS should be optimized for the PTM of interest. Further off-line fractionation might be needed to provide deep proteome coverage if PTM of interest is low in abundance.
Proteomics Data Analysis
40. Generate a concatenated target/decoy database from UniProt in Fasta format.
41. Generate MS2 files and search data using the ProLuCID algorithm in the Integrated Proteomics Pipeline (IP2) software platform. Basic searches were performed with the following search parameters: HCD fragmentation method; monoisotopic precursor ions; high resolution mode (3 isotopic peaks); precursor mass range 600-6,000 and initial fragment tolerance at 600 p.p.m.; enzyme cleavage specificity at C-terminal lysine and arginine residues with 3 missed cleavage sites permitted; static modification of +57.02146 on cysteine (carboxyamidomethylation), +229.1629 on N-terminal and lysine for TMT-10-plex tag; 4 total differential modification sites per peptide, including oxidized methionine (+15.9949), and phosphorylation (+79.9663) on serine, threonine, and tyrosine (only for phospho-enriched samples); primary scoring type by XCorr and secondary by Zscore; minimum peptide length of six residues with a candidate peptide threshold of 500; minimum of one peptide per protein and half-tryptic peptide specificity; Δmass cutoff = 10 p.p.m. with modstat, and trypstat settings; false-discovery rates of peptide (sfp) at 1%. Perform TMT quantification using the isobaric labeling 10-plex labeling algorithm, with a mass tolerance of 5.0 p.p.m. or less in cases where co-eluting peptide interfere. Use reporter ions 126.127726, 127.124761, 127.131081, 128.128116, 128.134436, 129.131417, 129.13779, 130.134825, 130.141145, and 131.13838 for relative quantification.
Melting Curve Analyses
42. Export peptide quantitative results from IP2 in Excel format.
43. Organize results in excel into 15 columns: one column displaying sequence peptide, one column displaying the custom identifier (Gene_pSite) that localizes the phosphosite in the protein, one column displaying number of spectra detected for that peptide sequence, 10 columns displaying TMT reporter ion intensities of the 10 different temperature fractions, one column displaying protein accession, and one column displaying protein description.
44. Use the consolidate function in Excel, integrate TMT reporter ion intensities of the same temperature fraction for peptides that map to the same phosphosite(s) using Gene_pSite column as filter.
45. Calculate fold change values by dividing all 10 reporter ion intensities by the reporter ion intensity of the 37°C fraction.
46. Generate unmodified protein and phosphomodiform melt curves, fit relative fold changes as a function of temperature to the equation derived from the chemical denaturation theory using the R package developed by Savitski et al (Science, 2014). Tmvalues are calculated at which the sigmoidal curve crosses the 0.5-fold change level.
47. Remove Tmvalues from curves with sigmoidal curve R2< 0.8.
48. Calculate ΔTmby subtracting Tmof the bulk, unmodified protein from that of the phosphomodiform. ΔTms could only be calculated for phosphomodiforms belonging to proteins that are also detected in the unmodified proteome.