Peptide library synthesis
Two singly-phosphorylated libraries, each consisting of 960 peptides, were synthesized by Cell Signaling Technology (Danvers, MA). The libraries were based on the following sequences Library A: [LREA][FKDG][TS]GH[PRDAF][EKG][st]LER and Library B: [LREA][FKDG][ts]GH[PRDAF][EKG][ST]LER, where lower case s/t indicate the site of phosphorylation. Contained within these libraries is the peptide used by Palumbo and Reid1 as an example showing evidence for gas-phase rearrangement (LFtGHPESLER). Other entries in the libraries were based on this general peptide scheme.
Yeast whole cell lysate phosphoproteome analysis
Wild type Saccharomyces cerevisiae
(BY4742) was used as a model organism for generating a complex mixture of unmodified and modified proteins. Yeast lysate was proteolyzed and fractionated as described previously10
. Cells were grown to mid-log phase (OD600
~ 0.45) in minimal media and collected by centrifugation at 4°C for 10 min, washed with cold water and re-centrifuged. The cell pellet was frozen and disrupted by bead beating in lysis buffer (2 × 90 sec bursts at 4°C). Lysis buffer consisted of 8M urea, 75 mM NaCl, 50mM Tris, pH 8.2, two tablets of protease inhibitor cocktail (complete mini, Roche) per 10 mL of lysis buffer, 50mM NaF, 50mM β-glycerophosphate, 1mM sodium orthovanadate, 10mM sodium pyrophosphate, 1 mM phenylmethanesulphonylfluoride (PMSF).
Cell lysate was assayed for protein concentration by the Bradford method. Ten mg of protein lysate was then reduced for 25 min at 56°C (with 5 mM dithiothreitol), alkylated at room temperature in the dark (in 14 mM iodoacetamide), and quenched with 5 mM dithiothreitol. The mixture was diluted with water to 4M Urea. Proteins were digested overnight at 37°C with 40 µg endoproteinase Lys-C. The sample was centrifuged and the supernatant was collected and lyophilized.
Lyophilized peptides were reconstituted with 1 mL 0.1% trifluoroacetic acid for desalting on a 200 mg tC18 SepPak cartridge (Waters). The cartridge was conditioned with 3 mL acetonitrile, 1 mL (7:3 acetonitrile:0.5% acetic acid), 1 mL (4:6 acetonitrile: 0.5% acetic acid), 3 mL 0.1% trifluoroacetic acid. The peptide mixture was applied to the column. Salts and impurities were washed with 3 mL 0.1% trifluoroacetic acid and 0.25 mL 0.5% acetic acid. Peptides were then eluted into a clean Eppendorf tube with 0.8 mL of 4:6 acetonitrile: 0.5% acetic acid and lyophilized.
Peptides were then fractionated by strong cation exchange (SCX) on a semi-preparative HPLC column (polySULFOETHYL A, 9.4-mm inner diameter 200 mm length, 5-mm particle size, 200 Å pore size (PolyLC)). Mobile phases consisted of (A) 7 mM KH2PO4, pH 2.65, 30% Acetonitrile (vol/vol); (B) 7 mM KH2PO4, 350 mM KCl, pH 2.65, 30% Acetonitrile (vol/vol); (C) 50 mM K2HPO4, 500 mM NaCl, pH 7.5. A gradient from 0% to 35% solvent B over 35 min, followed by a 5 min flush with solvent C, was used to fractionate the peptides. Fifteen fractions spanning the length of the gradient were collected and lyophilized. Peptides were again desalted on Sep Pak cartridges as described above but with 50 mg of tC18 material rather than 200 mg. Volumes of conditioning, loading, washing and elution buffer were adjusted accordingly, and fractions were again lyophilized.
Phosphopeptides were enriched over IMAC resin (Sigma, St. Louis MI) and desalted on C18 Empore 3M (3M, St. Paul, MN) stage tips. Each SCX fraction was resuspended in 120 µL of IMAC binding buffer (25 mM formic acid, 40 % acetonitrile) and incubated in 10 µL of a 50% slurry of IMAC beads for 1 hour at 21°C. The slurry was then poured over the stage tip previously condition with 20 µL of methanol, 20 µL of 50% acetonitrile, 0.5% acetic acid and 2 × 20 µL of 1% formic acid.
Non-phosphopeptides were eluted with 2×50µL IMAC binding buffer. The stage tip was equilibrated with 40 µL of 1% formic acid. Phosphopeptides bound to the IMAC resin were eluted twice with 70 µL of 500 mM K2HPO4, pH 7 and retained on the C18 Empore material. Phosphate salts were removed with 40 µL of 1% formic acid. Enriched phosphopeptides were eluted directly into HPLC vial inserts with 40 µL of 50% acetonitrile 0.5% acetic acid. Each fraction of enriched phosphopeptides was lyophilized and resuspended with 10 µL of 5% formic acid for LC-MS analysis.
Data were collected using an LTQ Orbitrap XL mass spectrometer with an additional ETD ion source, coupled to an LC system. Column tips were hand-pulled from 150 µm ID fused silica capillary. Microcapillary LC columns were prepared by packing 15 cm of C18 reverse-phase material (Magic C18AQ, Michrom BioResources, Auburn CA). A Famos autosampler (Dionex) with 10 µL loop and 2.4-µL injection needle was plumbed to two Agilent 1100 pumps operating between 80 and 300 µL/min and setup with a flow restrictor to provide an in-column flow rate of 0.5–1 µL/min. The LC program consisted of a 120-min method with 15-min loading time, 83.5-min gradient from 3% to 25% mobile phase B and 100 min of MS data collection. For each scan cycle, one high resolution survey scan acquired in the Orbitrap mass analyzer at 60,000 resolution, 106 automatic gain control (AGC) target and 1,000 ms maximum ion accumulation time, was followed by linear ion trap (LTQ) product ion scans (back-to-back CID and ETD). The 5 most intense precursor ions of appropriate charge state and minimum signal threshold of 3,000 were selected for fragmentation. CID scans were obtained with 120 ms maximum accumulation time, 2.0 Da isolation width and 30 ms activation at 29% normalized collision energy. ETD scans were obtained with 120 ms maximum accumulation time, 2.0 Da isolation width and 60 ms activation. Data were collected with a dynamic exclusion duration of 20 s. Precursor ions with a charge of 1+ or unassigned charge states were excluded.
Database searches and data filtering
All searches were performed using Sequest. For phosphopeptide libraries, an artificial database with 960 entries was assembled and reversed for target/decoy filtering to less than 0.5% false discovery rate (FDR) with multiple parameters including XCorr, dCN, and ppm. Filtered data from the two libraries were used to assess site assignment error rates associated with intramolecular phosphate transfer.
Two methods were used to assign phosphorylation sites. Sequest alone makes an attempt to localize modification sites. In addition, we employ Ascore, an algorithm for probability based phosphosite localization11
. Ascore exclusively considers site-determining ions for each STY and assigns modification site(s) based on the difference between site-determining ions for alternate possibilities. If the difference between the number of site determining ions is statistically large, then one can confidently assign the modification site. Notably, not all sites are confidently assigned with p-values < 0.05 (Ascore <13). However, a typical large scale phosphoproteomic study yields thousands of confidently assigned sites12
Raw data availability
All raw data are available at the Tranche web site (https://trancheproject.org
) including .RAW files (6) from triplicate analyses of Library A and Library B using back-to-back ETD/CID and 15 .RAW files for the yeast experiment. Peptide identifications from the libraries and yeast experiment with hyperlinks are available as Supplementary Table 1
and Supplementary Table 2