BURKHART et al THE QUANTITATIVE HUMAN PLATELET PROTEOME, SUPPLEMENT Supplemental Methods Sodium di-hydrogen phosphate (NaH2PO4), di-sodium hydrogen phosphate (Na2HPO4), sodium chloride and calcium chloride were purchased from Merck. Dithiothretiol (DTT) and Complete Mini protease inhibitor were acquired from Roche Diagnostics, bicichinonic acid (BCA) assay from Pierce, DryStrip cover fluid and Spec C18AR tips from Agilent. iTRAQ was obtained from Applied Biosystems, TiO2 beads/tips from GL Science, Immobiline DryStrips (pH 3-10) from GE Healthcare and HPLC solvents from Biosolve. The leukocount kit was obtained from BD Biosciences, all other chemicals from Sigma Aldrich. 1. Lysis, sample preparation and digestion Platelets were resuspended and lysed in 450 µl Gu-HCl, 50 mM Tris, pH 7.8 containing Complete mini, protein concentrations were determined by BCA. With exception of cysteine COFRADIC samples, disulfide bonds were reduced by addition of 10 mM DTT for 30 min at 56°C and free sulfhydryl groups were carbamidomethylated by adding 30 mM IAA for 30 min in the dark at room temperature. Cys-COFRADIC samples were prepared as described1. GuHCl was diluted to a concentration of 0.2 M using 50 mM NH 4HCO3 and samples were incubated with trypsin (protease:protein 1:20) overnight at 37°C. Samples were desalted by C18 solid phase extraction. 2. iTRAQ samples and fractionation To reduce sample complexity and increase proteome coverage and the number of quantitative data points, iTRAQ-labeled samples were subjected to a variety of prefractionation strategies, namely Met-COFRADIC2, Cys-COFRADIC1, strong cation exchange chromatography (SCX), hydrophilic interaction liquid chromatography (HILIC) and in-solution isoelectric focussing (IEF). Samples for label free (LF) estimation of copy numbers were fractionated by IEF, whereas samples for assessing intra-subject variation as well as TiO2 enriched samples were directly analyzed via LC-MS without prior fractionation. Prior to iTRAQ labeling samples were desalted, lyophilized and resuspended in 0.5 M TEAB according to the manufacturer’s instructions. To avoid label-biases, labels were switched 1 BURKHART et al THE QUANTITATIVE HUMAN PLATELET PROTEOME, SUPPLEMENT between different experiments. For label-free estimation of protein copy numbers (LF) and for TiO2 enrichment, 50 µg and 250 µg of digest per donor were pooled, respectively, and desalted. Finally, all samples were resuspended in the respective buffers for downstream processing. 2.1. COFRADIC reversed phase chromatography COFRADIC primary and secondary separations were performed on an Ultimate 3000 liquid chromatography system consisting of a LPG 3000 quaternary micro pump, a TCC 3400 column compartment, an UVD 3400 variable wavelength detector and a WPS-T 3000 well plate sampler equipped with 8-port valve, 0.15 mm bore for fractionation option, with a 125 µl loop (all Dionex, Germany). Labeled peptides were separated on a Zorbax 300SB-C18 column (5 µm particle size, 2.1 x 150 mm, Agilent) using a binary gradient (solvent A: 0.1% TFA; solvent B 0.08% TFA, 84% ACN) ranging from 5-80% B in 80min at a flow rate of 80 µL/min. 2.2. Methionine COFRADIC Upon desalting the iTRAQ labeled sample was resuspended in 0.1% TFA and separated by a primary RP-HPLC run. In total 48 fractions were collected minute by minute during the gradient, starting at 10% B. Fractions were pooled to 9 samples and methionine residues were oxidized prior to the secondary run for 30 min at 30°C by adding H 2O2 to a final concentration of 0.5% (v/v). Subsequently, each pooled sample was separated by a secondary RP-HPLC run, using identical chromatographic conditions. Due to the hydrophilic shift introduced by oxidation, Met containing peptides elute roughly 1-8 min earlier than in the primary run. The collected fractions were dried and prepared for LC-MS/MS analysis. 2.3. Cysteine COFRADIC The iTRAQ labeled sample was reconstituted in 0.1% TFA, H2O2 was added to a final concentration of 0.5% (v/v) followed by incubation at 30°C for 30 min. The sample was then separated in a primary RP-HPLC run. In total 48 fractions were collected during the gradient, starting at 10% B. Fractions were pooled to a final number of 16 fractions and subsequently dried under vacuum. Samples were resuspended in 10 mM Tris and incubated with 50 mM 2 BURKHART et al THE QUANTITATIVE HUMAN PLATELET PROTEOME, SUPPLEMENT DTT at 56°C for 30 min in order to remove DNTB. The reaction was stopped by adding 0.1% TFA (pH 2). All fractions were applied to a secondary RP-HPLC run, using identical chromatographic conditions. Fractions were recollected in a time interval 3 to 10 min before the respective elution time in the primary run. Collected fractions were dried under vacuum and prepared for LC-MS/MS analysis. 2.4. Strong cation exchange chromatography SCX was performed using a 1mmx15cm PolySULFOETHYL column (200 Å pore size, 5µm particle size, PolyLC, USA) in combination with an inert Ultimate HPLC system. The iTRAQ sample was resuspended in SCX buffer A and separated using a binary gradient (SCX buffer A: 5 mM NaH2PO4, pH 2.7; SCX buffer B: 5 mM NaH2PO4, 350 mM NaCl, 15% ACN, pH 2.7) ranging from 0 to 60% B in 45 min at a flow rate of 100 µL/min. Fractions were collected minute-by-minute, dried under vacuum and prepared for LC-MS/MS analysis. 2.5. Hydrophilic Interaction Liquid chromatography HILIC was performed using an inert Ultimate HPLC system (Dionex, Germering, Germany) with a zwitterionic ZIC® -HILIC stationary phase (SeQuant, Umeå, Sweden). For chromatographic separation a binary system (solvent A: 90% ACN, 10% ammonium acetate; solvent B: 40% ACN, 60% ammonium acetate) conducting a gradient elution from 0-100% B in 90 min. The iTRAQ sample was resuspended in solvent A and initially preconcentrated on a ZIC® -HILIC trapping column (240 µm x 4 cm) and subsequently separated on a ZIC-HILIC separating column (240 µm x 15 cm). Fractions were collected every two minutes. 3. Isoelectric focussing IEF was accomplished using an Agilent 3100 OFFGEL Fractionator (Agilent, Germany). For separation Immobiline DryStrips (GE Healthcare), pH 3-10, 24 cm length were used as described previously3. Briefly, IPG strips and samples were treated with rehydration solution containing 12% glycerol and ampholyte, pH 3-10 for 15 min. 150 µL of peptide solution were loaded into each well. For IEF, voltage was increased from 300 V to 8000 V within 12 h and 3 BURKHART et al THE QUANTITATIVE HUMAN PLATELET PROTEOME, SUPPLEMENT kept at 8000 V until a total of 64 kVh was reached. Finally fractions were desalted, evaporated and prepared for LC-MS/MS analysis. 4. TiO2 enrichment 250 µg platelet digest per donor were pooled, followed by a two-step TiO2 enrichment comprising a first TiO2 step with low and a second step with high phosphopeptide specificity, in order to improve the general proteome coverage. Therefore, self-packed (described previously by Beck et al.4) and commercially available TiO2 tips (GL Science) were used. Following C18-based desalting the sample was resuspended in 100 µL loading buffer (80% ACN, 6% TFA, saturated with phthalic acid) and incubated with TiO2 beads in a sample:bead ratio of 1:4 at RT for 30 min while gently shaking. After several washing steps using 100 µl of loading buffer and wash solution (80% ACN, 0.1% TFA), respectively, peptides were eluted successively using three buffers: (a) 0.5% NH4OH, 250 mM NH4HCO3, (b) 4.5% NH4OH, 0.3% H3PO4, 125 mM NH4HCO3, (c) 1.7% NH4OH and immediately acidified to pH 3 using TFA. Peptides which did not bind during the first loading step were enriched a second time with fresh beads. Eluates were combined, desalted using Omix C18 tips, an aliquot was measured by LC-MS/MS and the rest subjected to a second TiO2 step in order to yield a high phosphopeptide-specificity. 5. Mass spectrometry iTRAQ Samples were analyzed on a QstarElite (Applied Biosystems) and an LTQ-Orbitrap XL (Thermo Scientific) online coupled to U3000 nano-HPLC systems (Dionex). Peptides were preconcentrated on an in-house packed 100 µm inner diameter C18 trapping column (Synergi HydroRP, Phenomenex, 4 µm particle size, 80 Å pore size, 2 cm length) in 0.1% trifluoroacetic acid and separated on an in-house packed 75 μm inner diameter C18 maincolumn (Synergi HydroRP, Phenomenex, 2 µm particle size, 80 Å pore size, 30 cm length) applying a binary gradient from 4-42% acetonitrile in 0.1% formic acid. On the Orbitrap XL, MS survey scans were acquired from 300-2,000 m/z at a resolution of 60,000 using the polysiloxane m/z 445.120030 as lock mass5 and the three most intense ions (Top3) were first 4 BURKHART et al THE QUANTITATIVE HUMAN PLATELET PROTEOME, SUPPLEMENT analyzed by collision induced dissociation (CID)-based MS/MS in the LTQ, followed by higher energy collision induced dissociation (HCD)-based MS/MS in the Orbitrap (resolution 7,500). AGC values were set to 104 for MS/MS and to 106 for MS scans. On the Qstar Elite, survey scans were analyzed from 350-2,000 m/z and the Top3 were selected for MS/MS with the iTRAQ reagent option, the fragment intensity multiplier set to 20 and a maximum accumulation time of 2 s. LF IEF fractions were analyzed on an LTQ Orbitrap Velos, essentially as described above, analyzing the Top20 by CID-MS/MS in the ion trap. TiO2 enriched samples were analyzed on a q-Exactive as described above, Top15. AGC values were set to 2·105 for MS and 105 for MS/MS scans. Intra-variation samples were analyzed on a q-Exactive mass spectrometer coupled to an Ultimate 3000 RSLC system (Thermo Scientific), using a 75 µm x 2 cm C18 trapping column and a 75 µm x 50 cm C18 main column (both Pepmap, Thermo Scientific) and a 3 h LC gradient ranging from 3-40%B at 60°C. 6. Peak list generation All raw file were converted into mzML6 using msconvert as part of the Proteowizard 1.6.0 package7. Peak lists in mgf format were subsequently obtained using OpenMS version 1.8 8. (A) Orbitrap Velos files were converted using the FileConverter; (B) Orbitrap XL files were separated according to the fragmentation method, OpenMS high resolution peak-picker was then applied on HCD spectra, for each LC-MS analysis, resulting peak lists (CID and HCD) were then grouped in one mgf file; (C) Qstar Elite spectra were first processed with a BaselineFilter in order to correct for baseline deviations, subsequently peak-picked using OpenMS high resolution peak-picker and then converted into mgf format; (D) q-Exactive spectra were peak-picked using OpenMS high resolution peak-picker and transformed into mgf format. 7. Database searches Spectra were searched using multiple search engines in order to improve proteome coverage9, namely Mascot10 v2.3, OMSSA11 v2.1.9 and X!Tandem12 v2010.01.01 using SearchGUI13 and the settings listed in supplemental table 1. All searches were searched 5 BURKHART et al THE QUANTITATIVE HUMAN PLATELET PROTEOME, SUPPLEMENT against a concatenated target/decoy version of the UniprotKB/SwissProt human database (4th of November 2010; 20,260 target sequences), generated using dbtoolkit14, allowing to estimate the amount of retained false positive identifcations15. Parameters used for searching are listed in supplemental table 1. Identification results were extracted from search engine result files using MascotDatfile parser16, OMSSA parser17 and X!Tandem parser18. Spectra matching peptide sequences that were shared between target and decoy databases were omitted. To reduce the amount of false positive identifications, PSMs meeting the following criteria were removed19,20: (1) sequences <8 and >20 amino acids, (2) e-values >10, (3) precursor mass deviations >10 ppm (Orbitrap) and >0.05 Da (Qstar). 8. Identification results post-processing Results obtained from Mascot, OMSSA and X!Tandem were imported into PeptideShaker (http://peptide-shaker.googlecode.com) excluding hits with an e-value over 10, peptide length under 8 or over 20 amino acids and precursor mass deviations over 10 ppm (Orbitrap, q-Exactive, Velos) or 0.05 Da (QStar Elite). Note that PeptideShaker automatically excludes peptides deriving from both the target and the decoy database. The following steps were subsequently conducted: (1) normalization of Peptide to Spectrum Matches (PSMs) using Posterior Error Probabilities (PEPs), (2) merging of search engine results, (3) Peptide inference and scoring, (4) Protein inference and scoring and (5) validation at 1% False Discovery Rate (FDR). 9. Estimation of Posterior Error Probabilities A list of all first hits was generated for each search engine and sorted by e-value. PEPs were estimated for each PSM by comparing the decoy distribution to the target distribution with a binning of Nmax, the maximal amount of target hits comprised between two subsequent decoy hits20. The amount of decoy hits Ndecoy was used as an estimate for the amount of false positives thus allowing the straightforward estimation of the Posterior Error Probability (PEP) for each match by: 6 BURKHART et al THE QUANTITATIVE HUMAN PLATELET PROTEOME, SUPPLEMENT Μ= ππΈπ ππππππ¦ ππππ₯ In the results presented in this study a confidence is estimated for each match straightforwardly defined as ππππππππππ = 1 − ππΈπ. 10. Search engine results unification For each MS/MS spectrum, the probability that all search engines p spectrum were wrong was estimated as a product of all search engine PEPs: Μ Μ Μ ππ ππππ‘ππ’π Μ = ππΈπ πππ πππ‘ . ππΈππππππ΄ . ππΈππ!ππππππ A low pspectrum thus suggests that at least one search engine assigned the right peptide to the spectrum confidently. In case of search engine disagreement, the most likely peptide (i.e. the lowest PSM PEP) was retained. The spectra were sorted according to ππ ππππ‘ππ’π Μ and the PEP of the retained PSM was estimated as described above. Note that PSMs were grouped according to the identified charge when statistical significance was ensured. We considered that statistical significance was ensured for a charge state when its Nmax was higher than 100 and more than 100 PSMs scored better than the first decoy hit. 11. Establishment of a peptide list PSMs were combined into all possible peptides. It is important to note here that differentially modified peptides were distinguished yet the modification site-localization was not taken into account as its assessment is not possible for all spectra. As it is known that the peptide PEP grows with the product of the PSM PEPs 9, peptides were scored using this metric: π Μ π ππππππππ‘πππ = ∏ ππΈπ ππππ π=1 Μ where N represents the amounts of PSMs identifying the peptide of interest and ππΈπ ππππ the estimated PEP of the ith PSM. The PEP of each peptide is yet not equal to this score as already demonstrated21 and was thus re-estimated using the above method. Note that 7 BURKHART et al THE QUANTITATIVE HUMAN PLATELET PROTEOME, SUPPLEMENT peptides with different modifications were processed separately if statistical significance was ensured. 12. Establishment of a protein list Following the same rationale proteins were scored using the product of peptide PEPs: π π ππππππππ‘πππ = ∏ ππΈπΜ ππππ‘ππππ π=1 th Where ππΈπΜ ππππ‘ππππ represents the estimated PEP of the i peptide among the N identifying this protein. Here it is crucial to note the presence of shared peptides between various proteins. In order to prevent the statistics from being impaired by these so-called degenerated peptides, groups of proteins were generated as prescribed by Nesvizhskii 9: Protein A Protein B Peptide 1 Peptide 2 Peptide 3 Peptide 4 As illustrated in figure 1, in case that 4 peptides are shared between two proteins, three protein matches will be generated with three scores: protein A from peptide 1 with π ππππππππ‘πππ π΄ = ππΈπΜ ππππ‘πππ 1 ; protein AB from peptide 2 with π ππππππππ‘πππ π΄π΅ = ππΈπΜ ππππ‘πππ 2 ; protein B from peptides 3 and 4 with π ππππππππ‘πππ π΅ = ππΈπππππ‘πππΜ 3 . ππΈπππππ‘πππ 4 . In this model peptides are thus unique to protein groups allowing the estimation of the PEP of every protein group. In the following, a protein can thus implicitly denominate a group of proteins. Based on the previous example, when sorting protein matches based on their confidence, three cases can occur: (1) both matches A and B are scoring better than match AB; (2) A or B is scoring better than AB; (3) neither A neither B is scoring better than AB. In cases (1) and (2) the match AB is removed as sufficient peptide evidence allows the validation of a single protein. In case (3) all cases are kept in the dataset potentially generating protein lists 8 BURKHART et al THE QUANTITATIVE HUMAN PLATELET PROTEOME, SUPPLEMENT including A and AB. Indeed, in this case we cannot discriminate A from B. A clear example in the presented results is the presence of various forms of Actin with extensive sequence similarities. Yet, sufficient unique peptides confidently identified allowed us to discard the intersection groups. 13. FDR validation The final PSM, peptide and protein sets were validated at 1% FDR, indicating that the expected result set contains one percent of false positives. For this, hits are sorted according to their respective score and the amount of false positives is estimated using the number of decoy hits. The highest score validating less decoy hits than 1% of the resulting dataset size is retained for hit validation. 14. Phosphorylation site scoring Although instruments allowed tremendous progress in PTM analysis, the location of phosphorylation remains often uncertain. In order to assess the validity of the phosphorylation site returned by the identification process, we combined the use of two well-known PTM scoring approaches: the A-score19 and the scoring through secondary hit scores4. For the later, the search engine independent score attached to the phosphorylation site is the difference between the next hit with the first secondary hit with the same sequence yet another phosphorylation site and the best hit PEP. Concretely, it corresponds to the confidence increase when going from the second best location to the best according to the search engines result. The A-score was estimated for singly modified peptides only. A phosphorylation site was marked confident when both scores did agree on the phosphorylation site or when the PEP difference was above 0.50. 9 BURKHART et al THE QUANTITATIVE HUMAN PLATELET PROTEOME, SUPPLEMENT 15. Absolute quantification Proteins were quantified using the Normalized Spectrum Abundance Factor 22 (NSAF). Briefly, here the number of validated spectra which can be ascribed to a protein match is divided by the length of the protein. 16. Relative quantification After discarding spectra containing null intensities, iTRAQ intensities were extracted from mgf files and deisotoped using the isotope matrix provided by the manufacturer 23. PSM iTRAQ ratios were calculated by dividing reporter intensites 115, 116 and 117 m/z by the 114 reporter intensity. PSM ratios were used to compute peptide and finally protein ratios. When less than six PSM (respectively peptide) ratios were available for peptide (respectively protein) ratio estimation, the median of ratios was taken. Otherwise the peptide (respectively protein) ratio was estimated using a redescending M-estimator chosen for its robustness and accuracy24. In order to compensate for systematic errors, the log of each reporter ratio was normalized to the median of all the respective ratios. For every protein, the four ratios (114/114, 115/114, 116/114 and 117/114) were subsequently normalized by the median of the ratios. 17. Comparison of proteome and transcriptome data Protein assignment was conducted (http://www.uniprot.org/?tab=mapping) using and PICR the Uniprot mapping tool (http://www.ebi.ac.uk/Tools/picr/). Identifiers which could not be assigned to a protein were searched in the NCBI database (http://www.ncbi.nlm.nih.gov/sites/batchentrez). Obsolete/deleted entries were blasted (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and manually assigned. Entries referring to pseudogenes or non-coding RNA were removed, except for non-coding transcripts of proteins which were taken as indicators for protein expression. Protein assignments for proteome and transcriptome data were matched, merged, and entries unique to either dataset were matched to ambiguous entries in the other one. To improve significance of transcriptome data, a cutoff of one reads per kilobase of exon model per million mapped reads (RPKM) was 10 BURKHART et al THE QUANTITATIVE HUMAN PLATELET PROTEOME, SUPPLEMENT applied. Frequency analysis was conducted by classifying copy numbers in classes with logarithmic distances. A normal distribution function was approximated to the data. For comparison of frequencies the data were stratified into percentiles according to the frequency of copy numbers. 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