Supplementary Materials and Methods
Liquid Chromatography Mass Spectrometry. Sample preparation and in situ tryptic digestion were
performed as previously described.(1) Mass spectrometry analyses were on a quadrupole time-offlight (QTOF) mass spectrometer (QTOF Premier or QTOF Ultima, Waters, Manchester, UK) equipped
with a nanoelectrospray ion source coupled to a high-performance liquid chromatography Agilent 1100
nanoflow system (Agilent Technologies, Palo Alto, CA). The HPLC system was comprised of a solvent
degasser, column compartment, micro well-plate autosampler, binary pump and nanoflow pump.
Solvent A consisted of 0.4% acetic acid, 0.005% heptafluorobutyric acid (HFBA) in water and solvent
B consisted of 0.4% acetic acid, 0.005% HFBA in 90% acetonitrile. From a thermostatted
microautosampler, 8 µL of the tryptic peptide mixture were automatically loaded onto a trap column
(Zorbax 300SB-C18 5 µm 5×0.3 mm, Agilent Technologies, Palo Alto, CA) with the binary pump at a
flow rate of 40 µL/min (100% solvent A). After the trap column was washed with 100% solvent A, the
peptides were back-flushed onto a customized 16 cm fused silica analytical column (inner diameter 50
µm) packed with C18 reversed-phase material (ReproSil-Pur 120 C18-AQ, 3µm, Dr. Maisch GmbH,
Ammerbuch-Entringen, Germany). The peptides were eluted from the analytical column with a 4
minute gradient ranging from 3 to 13 percent solvent B, followed by a 35 minute gradient from 13 to 35
percent solvent B, followed by an 11 minute gradient from 35 to 50 percent solvent B and, finally, a 6
minute gradient from 50 to 100 percent solvent B at a constant flow rate of 80 nL/min. The analysis
was performed in a data-dependent acquisition mode using one MS channel for every three MSMS
channels and a dynamic exclusion for selected ions of 60 s (MassLynx, Waters, Manchester, UK).
Data Analysis. The acquired data were processed with ProteinLynx Global Server 2.2.1 (Waters,
Manchester, UK) and searched against the human IPI database version v3.32/41 with the search
engine MASCOT.(2, 3) Submission to MASCOT was via a Perl script that performs an initial search
with relatively broad mass tolerances on both the precursor and fragment ions (200 ppm and 0.15
Da, respectively). High-confidence peptide identifications are used to recalibrate all precursor and
fragment ion masses prior to a second search with narrower mass tolerances (15 ppm and 0.05 Da,
respectively). One missed tryptic cleavage site was allowed. Carbamidomethyl cysteine was set as a
fixed modification, and oxidised methionine was set as a variable modification. For the validation of
proteins, MASCOT output files were processed by an internally-developed parser whereby two unique
peptides with an ion score greater than, or equal to, 20 were required. The validated proteins were
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subsequently grouped according to shared peptides and a false positive detection rate of less than
0.5% was determined by applying the same procedure against a reversed database.
Protein
identifications from all 18 samples (affinity purifications) or all 50 duplicate samples (‘core
proteome’)(1, 4)
were combined to compute protein groups. Each protein group consists of one
anchor protein that has peptides identified by mass spectrometry that are specific for that protein plus
a subset of proteins that contain peptides also matching the anchor protein. That is, each protein
group represents a protein with supporting experimental evidence and proteins that do not have
specific experimental support are not considered.
References
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the kinase inhibitor bosutinib in primary chronic myeloid leukemia cells. Leukemia 2009; 23: 477-485.
2 Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based protein identification by searching
sequence databases using mass spectrometry data. Electrophoresis 1999; 20: 3551-3567.
3 Kersey PJ, Duarte J, Williams A, Karavidopoulou Y, Birney E, Apweiler R. The International Protein
Index: an integrated database for proteomics experiments. Proteomics 2004; 4: 1985-1988.
4 Schirle M, Heurtier M-A, Kuster B. Profiling core proteomes of human cell lines by one-dimensional
PAGE and liquid chromatography-tandem mass spectrometry. Mol Cell 2003; 2: 1297-1305.
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