SUPPLEMENTARY METHODS
Expression of NTAP(GS)- IKK in E.coli and purification by affinity chromatography
E.coli (strain BL21 DE3) were transformed with pETM30-NTAP(GS)-IKK 1. This plasmid
encodes a fusion protein of NTAP(GS)-IKK with GST and with a HexaHis tag (both located
at the N-terminus: HexaHis-GST-NTAP(GS)-IKK). The fusion protein was purified by a
single step using HisTrap columns (Amersham). The purified HexaHis-GST-NTAP(GS)-IKK
was desalted using a HiPrep desalting column (Amersham) and stored at -80°C. A fraction of
the obtained HexaHis-GST-NTAP(GS)-IKK was subjected to TEV cleavage. As both TEV
and the N-terminal cleavage products contain a His tag, these contaminants were removed
by incubation with Ni-NTA agarose. The supernatant contains the purified IKK devoid of the
TAP-tag.
Sample Preparation
TAP samples were alkylated with iodoacetamide and separated by 1D SDS-PAGE on a 4 –
12% bis-Tris gel (NuPAGE, Invitrogen, CA).
After visualisation of the proteins by silver
staining, specific bands and/or regions of interest were excised from the gel and digested in
situ with modified porcine trypsin (Promega Corp., Madison, WI) essentially as described by
Shevchenko et al 2. Prior to analysis by nanoLC-MSMS, tryptically-digested samples were
purified and concentrated via customised reversed-phase columns adapted from Mann et al
3
.
Mass Spectrometry and Data Analysis
Tryptically-digested samples were analysed by data-dependent nanocapillary reversedphase LC-MSMS using customised 75 µm inner diameter columns packed with C18 3 µm
diameter Reprosil beads (Maisch, Germany) on a nanoLC system (Agilent Technologies,
Palo Alto, CA) coupled to a quadrupole time-of-flight (QTOF) mass spectrometer (QTOF
Ultima, Waters, UK). Direct injection data-dependent acquisition was performed for 75 min
using one MS channel for every three MSMS channels and a dynamic exclusion for selected
ions of 60 s.
Proteins were identified by automated database searching (Mascot Daemon, Matrix Science,
London, UK) against an internally-curated version of the monthly updated International
Protein Index protein sequence database (IPI, versions 3.14 and 3.15, European
Bioinformatics Institute, www.ebi.ac.uk/IPI/). This compilation of entries from Swiss-Prot,
TrEMBL, RefSeq, and Ensembl was appended with frequently-observed non-human
contaminants and viral proteins expressed by the immortalised HEK293 cell line, e.g., E1B
protein from human adenovirus type 5 (NCBInr accession number gi|56160532). Search
parameters were as follows: MS and MSMS tolerance of 0.1 Da, tryptic specificity allowing
for 1 missed cleavage site and K/R cleavages (accounts for in-source fragmentation of tryptic
peptides followed by a proline residue), fixed modification of carbamidomethylation of
cysteine residues, and variable modification of oxidation of methionine residues. To assess
the incidence of false positive identifications, the data were also searched against a inverted
tryptic peptide database, i.e., a theoretical tryptic digest of the database was performed and
for each tryptic peptide the sequence of the peptide was reversed but still maintained a
tryptic peptide identity (inverted peptides terminate in a lysine or arginine residue). Data
indicated that the false positive rate was <0.9%. Results from the database search were
parsed into EPICenter (Proxeon Biosystems, Odense, Denmark) for automated validation
and protein grouping based on the number of shared peptides identified by MSMS 4.
Criterion for a positive protein identification was identification of a minimum of 2 peptides with
a Mascot peptide score of >= 20. Data comparison and data filtering were also performed
using EPICenter.
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4.
Hantschel, O. et al. Structural basis for the cytoskeletal association of Bcr-Abl/c-Abl.
Mol Cell 19, 461-473 (2005).
Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of
proteins silver-stained polyacrylamide gels. Anal Chem 68, 850-858 (1996).
Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrixassisted laser desorption/ionization, nanoelectrospray, and LC/MS sample
pretreatment in proteomics. Anal Chem 75, 663-670 (2003).
Kristensen, D.B. et al. Experimental Peptide Identification Repository (EPIR): an
integrated peptide-centric platform for validation and mining of tandem mass
spectrometry data. Mol Cell Proteomics 3, 1023-1038 (2004).
2