Data Set 1 - Mast cell phosphopeptides (MCP5).  Transformed primary bone marrow-derived mast cells (MCP5) were stimulated as described previously.[1]  Stimulated cells (~5x108) were lysed for 10 min with rotation at 4°C in 50 mL of lysis buffer  consisting of 20 µg/mL aprotinin, 20 µg/mL leupeptin, 50 mM Tris pH 7.5, 100 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM Perfabloc, 2 mM Na3VO4, 10 mM β-glycerophosphate and 1 mM EDTA (all from Sigma, St. Louis, MO).

Lysates were centrifuged at 12,000x g for 20 minutes at 4°C.  Fifty pmol of LIEDAEpYTAK per ~5x108 cell equivalents (c.eq.) and anti-phosphotyrosine agarose (clone PT66, Sigma; 200 µL resin/~5x108 cell equivalents) was added to the supernatant for 4 hr at 4° C with rotation.  Beads were washed once with 50 mL lysis buffer and once with 50 mL of 20 mM Tris buffer, pH 7.4, 120 mM NaCl.  Proteins were recovered from the beads with 100 mM NH4HCO3 buffer pH 8.3 containing 8 M urea for five minutes at 96° C and the supernatant was filtered using PVDF membranes with a pore size of 0.22 µm (Millipore Inc., Bedford, MA).  The mixture was diluted with an equal volume of water and proteins were digested overnight with 5 µg of modified trypsin (Promega, Madison, WI) at 37° C.

Tryptic peptides were converted to methyl esters with deuteromethanol so that every Asp, Glu and peptide c termini contained an additional mass of 17 Da.  Phosphopeptides were enriched with an automated desalt/immobilized metal affinity chromatography (IMAC)/nano-liquid chromatography/ electrospray ionization mass spectrometry platform as described previously.[2]  Peptides where eluted with a 30 minute 0-70% solvent B reversed-phase gradient through an analytical column with integrated 4 µm electrospray tip into an LTQ mass spectrometer with 30 nl/min peak parking (solvent A - 0.1 M acetic acid ddH2O, solvent B - 0.1 M acetic acid acetonitrile).[2]  Peptides were analyzed by data dependent tandem mass spectrometry (MS/MS) experiments using collisionally-induced dissociation (Xcalibur 1.4 parameters designated 35% collision energy, 3 Da isolation window, top 5 data dependent, repeat count of one, and a dynamic exclusion time of 1.5 min, IT-MS AGC of 30,000, and IT-MS/MS AGC of 10,000).  MS/MS spectra were assigned to peptide sequences from the NCBI non-redundant protein database sliced in Bioworks 3.1 for human proteins and searched with the SEQUEST algorithm.  SEQUEST search paramaters designated a static modification of +17.0342 Da on Asp, Glu and the c-terminus (deuteromethyl esters) and variable modifications of +79.9663 Da on Ser, Thr, and Tyr (phosphorylation).

Data Set 2 - Pervanadate stimulated T cell phosphopeptides (PVIP).  Cell culture was performed as described.[3]  Briefly, Jurkat cells (clone E6-1) were grown in RPMI 1640 medium with 10% fetal bovine serum, 2 mM L-glutamine, 100 ug/ml streptomycin sulfate, and 100 U/ml penicillin G (all from Sigma) in a 5.0% CO2 incubator.  Cells were treated with pervanadate to inhibit tyrosine phosphatases and elevate levels of phosphotyrosine as described previously.[4]  Cells (1x109) were washed with RPMI lacking FBS at 4°C then lysed for 20 min with rotation at 4°C in 25 mL of lysis buffer.  Cellular lysates were purified and analyzed as in Data Set 1 with the exception that the amount of cell equivalents analyzed was reduced to 1x108 as compared with 5x108 for Data Set 1.  MS/MS spectra were assigned to peptide sequences from the NCBI non-redundant protein database sliced in Bioworks 3.1 for human proteins and searched with the SEQUEST algorithm.  SEQUEST search paramaters designated a static modification of +17.0342 Da on Asp, Glu and the c-terminus (deuteromethyl esters) and variable modifications of +79.9663 Da on Ser, Thr, and Tyr (phosphorylation).

Data Set 3 - Standard protein mixture (18Mix).  The publicly available raw data files acquired on LTQ instrument and the validated peptide assignments were downloaded from http://regis-web.systemsbiology.net/PublicDatasets/ [5] .  MS/MS spectra were assigned to peptide sequences from the NCBI non-redundant protein database sliced in Bioworks 3.1 for H. influenzae and searched with the SEQUEST algorithm.  SEQUEST search paramaters designated a static modification of +57.0215 Da on Cys (alkylation).

Data Set 4 - Bovine serum albumin peptides (BSA).  Bovine serum albumin (BSA; Sigma) was reconstituted in 8M urea, 100 mM ammonium bicarbonate, pH 8.0.  Tris-carboxyethyl phosphine was added to 10 mM and the mixture allowed to stand at room temperature for 10 minutes.  Iodoacetamide was then added to a final concentration of 20 mM, and the mixture was incubated at 22 ˚C for 45 min. in the dark.  The mixture was then diluted with an equal volume of 100 mM ammonium bicarbonate, pH 8.0.  Modified trypsin (Promega) was added, and the mixture incubated for 8 hr at 37 °C.  BSA peptides were enriched with our automated system as in Data Set 1 except the desalt and IMAC separations were skipped.[2]  Peptides where eluted with a 30 minute 0-70% solvent B reversed-phase gradient through an analytical column with integrated 4 µm electrospray tip into an LTQ mass spectrometer with no peak parking (solvent A - 0.1 M acetic acid, solvent B - 0.1 M acetic acid acetonitrile).[2]  Data acquisition parameters were identical to Data Set 1.  MS/MS spectra were assigned to peptide sequences from the bovine NCBI non-redundant protein database with the SEQUEST algorithm.  SEQUEST search parameters designated a static modification of +57.0215 Da on Cys (alkylation).

Data Set 5 – Insulin stimulated 3T3 phosphopeptides (3T3).  Cell culture was performed as described.[6] Briefly, 3T3 cells transfected with IRS-1 were incubated with DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 400 ug/ml G418 (all from Sigma except L-Glu which from Invitrogen) in a 5.0% CO2 incubator. After reaching 95% confluence, Cells were starved in DMEM with 0.1% BSA for 24 hours. On the next day, cells were stimulated with 100 nM insulin for 5 minutes and then immediately lysed in cold lysis buffer consisting of 8 M urea, 100 mM NH4HCO3 and 1 mM NaV3O4 for 20 minutes. The lysates were reduced, alkylated, digested, and desalted as described [1]. Purified peptides were then fractionated with a homemade SCX column (500 μm x 15 cm PEEK tubing (Upchurch, Oak Harbor, WA) packed with 5 μm PolyLC SCX resin (The Nest Group, Southborough, MA)). Phosphopeptides in each fraction were enriched by TiO2 as described [7] and analyzed on LC/MS as in Data Set 3. MS/MS spectra were assigned to peptide sequences from the human NCBI non-redundant protein database with the SEQUEST algorithm.  SEQUEST search parameters designated a static modification of +57.0215 Da on Cys (alkylation) and variable modifications of +79.9663 Da on Ser, Thr, and Tyr (phosphorylation).

 

 

Reference

 

[1] Cao, L., Yu, K., Banh, C., Nguyen, V., et al., J Immunol 2007, 179, 5864-5876.

[2] Ficarro, S. B., Salomon, A. R., Brill, L. M., Mason, D. E., et al., Rapid Commun Mass Spectrom 2005, 19, 57-71.

[3] Brill, L. M., Salomon, A. R., Ficarro, S. B., Mukherji, M., et al., Anal Chem 2004, 76, 2763-2772.

[4] Secrist, J. P., Burns, L. A., Karnitz, L., Koretzky, G. A., Abraham, R. T., The Journal of biological chemistry 1993, 268, 5886-5893.

[5] Klimek, J., Eddes, J. S., Hohmann, L., Jackson, J., et al., Journal of proteome research 2008, 7, 96-103.

[6] Tanaka, S., Ito, T., Wands, J. R., The Journal of biological chemistry 1996, 271, 14610-14616.

[7] Olsen, J. V., Blagoev, B., Gnad, F., Macek, B., et al., Cell 2006, 127, 635-648.