MS raw file processing

The output from one LC-MS/MS run from the above mentioned instruments is a raw file containing amongst others information about retention time, m/z values, intensities and the charge state of intact peptide precursors and product ions of selected precursors. In order to identify and quantify peptides, proteins, and PTM sites, the raw files were submitted to the software MaxQuant. MS/MS spectra were searched against a human proteome database with the search engine Andromeda which is implemented in MaxQuant (Cox and Mann, 2008; Cox et al., 2011). In the following paragraph, raw file processing for the different projects are described.

2.2.4.1. BL experiments

All raw files were processed using MaxQuant (Versions between 1.5.0.25 and 1.5.2.8) against a UniProtKB/Swiss-Prot human database containing 88,993 and 89,706 entries (downloaded July 2014 or January 2015), respectively (Cox and Mann, 2008). Raw files of the protein

expression, global phosphoproteome and pYome experiments and different cell lines were processed either independently from each other or categorically grouped e.g. all pYomes or all files of one cell line together. Cysteine carbamidomethylation was set as fixed modification, methionine oxidation and serine, threonine and tyrosine phosphorylation for both the pYome and the global phosphoproteome dataset as variable modification. The ‘Minimum Andromeda Score’ and ‘Delta score’ for modified peptides was set to 40 and 6, respectively. The following parameters were applied: the MS1 first search peptide tolerance was set to 20 ppm and the main search peptide tolerance to 4.5 ppm. The FTMS MS/MS tolerance was set to 20 ppm, a false discovery rate (FDR) of 1% for peptide spectrum matches (PSM), protein and site decoy was applied, 7 amino acids as minimum peptide length and 2 maximum missed cleavages were allowed. Multiplicity was set to two in a double or to three in a triple SILAC-based quantitation experiments (duplex label: Lys+0 Arg+0, Lys+8 Arg+10; additionally for triplex labeling Lys+4 Arg+6). The ‘Re-quantify’ option of MaxQuant was enabled. Unique, razor, unmodified, N-terminally acetylated and methionine oxidized peptides were used for protein quantitation and the minimum ratio count required was two. For label-free quantitation, the MaxLFQ algorithm was enabled with a LFQ min. ratio count two (Cox et al., 2014). Additionally, the

‘Match between runs’ function was enabled at default settings of a match time window of 0.7 min and an alignment time window of 20 min.

2.2.4.2. Kinobead analysis

Progenesis software (Version 3.1; Nonlinear Dynamics, Newcastle, UK) was used for intensity-based label-free quantitation. Briefly, after selecting one sample as a reference, the retention times of all eluting precursor m/z values in all other samples within the experiment were aligned creating a large list of ‘features’ representing the same peptide in each sample.

Features with two to five charges were included; features with two or less isotopes were excluded for further analysis. After alignment and feature filtering, raw abundances of all features were normalized to determine a global scaling factor for correcting experimental variation such as differences in the quantity of protein loaded into the instrument. Given that multiple MS/MS spectra are frequently collected for the same feature (precursor ion) across all the samples, the precursor intensities were ranked, and the MS/MS spectra of the five most intense precursors for each feature were transformed into peak lists and exported to generate Mascot generic files. The Mascot generic files were searched against the Uniprot human protein sequence database (Version 22.07.13, containing 88,354 entries) using Mascot

Materials and Methods

(Version 2.2, Matrix Science, London, UK). Search parameters were set as follows:

carbamidomethylation of cysteine residues as fixed modification, serine, threonine, and tyrosine phosphorylation and methionine oxidation as variable modification, trypsin as proteolytic enzyme with up to two miss cleavages, precursor ion mass tolerance of 10 ppm, fragment ion mass tolerance of 0.05 Da, decoy search enabled. Search results for spectrum to peptide matches were exported in xml format and then imported into Progenesis software to enable the combination of peptide quantitation and identification. Peptides with Mascot ion scores <25 were filtered out, and only unique peptides for corresponding proteins were used for identification and quantitation. For protein quantitation, the feature intensities of all unique peptides of a protein were summed up.

2.2.4.3. AML experiments

The protein expression and pYome raw files were analyzed with MaxQuant v1.3.0.5 and the GP was analyzed with MaxQuant v1.5.0.25 against a UniProtKB/Swiss-Prot human database containing 88,993 or 89,706 entries (downloaded July 2014 or January 2015). Raw files of KG1 and MV4-11 cells were processed together whereas protein expression, global phosphoproteome and pYome experiments were processed independently from each other.

Cysteine carbamidomethylation was set as fixed modification, methionine oxidation and serine, threonine and tyrosine phosphorylation for both the pYome and the global phosphoproteome datasets as variable modification. The ‘Minimum Andromeda Score’ and

‘Delta score’ for modified peptides was set to 40 and 6, respectively for the global phosphoproteome (analyzed with MaxQuant v1.5.0.25). The following parameters were applied: the MS1 first search peptide tolerance was set to 20 ppm and the main search peptide tolerance to 4.5 ppm for the global phosphoproteome (Protein expression and pYome were analyzed with MaxQuant v1.3.0.5: 6 ppm), respectively. The FTMS MS/MS tolerance was set to 20 ppm, a FDR of 1% for PSM/peptide, protein and site levels was applied. Seven amino acids as minimum peptide length and maximum two missed cleavages were allowed.

Multiplicity was set to two (duplex label: Lys+0 Arg+0, Lys+8 Arg+10). The ‘Re-quantify’ option of MaxQuant was enabled and the minimum ratio count required was set to two.

2.2.4.4. TCR signaling adapters

Raw files from four independent biological replicates were processed together as distinguishable experimental groups using MaxQuant (Version 1.5.2.8) against a

UniProtKB/Swiss-Prot human database containing 89,706 entries (downloaded January, 2015). Raw files of different biological replicates were defined in experimental groups and different proteinase specificities were defined in parameter groups. Cysteine carbamidomethylation was set as fixed modification, methionine oxidation and serine, threonine and tyrosine phosphorylation as variable modification. The ‘Minimum Andromeda Score’ and ‘Delta score’ for modified peptides was set to 40 and 6, respectively. The following parameters were applied: the MS1 first search peptide tolerance was set to 20 ppm and the main search peptide tolerance to 4.5 ppm, respectively. The FTMS MS/MS tolerance was set to 20 ppm, the decoy mode was set to revert, a FDR of 1% for PSM, protein and phosphorylation site was applied, seven amino acids as minimum peptide length and maximum two missed cleavages were allowed. Multiplicity was set to two: Lys+0 Arg+0 Tyr+0 and Lys+8 Arg+10 Tyr+10. Unique, razor, unmodified, N-terminally acetylated and methionine oxidized peptides were used for protein quantitation and the minimum ratio count required was two. The ‘Re-quantify’ option of MaxQuant was enabled.