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Supplemental Information
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Materials and Methods
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Standard laboratory reagents were purchased from Sigma, Roth and Merck unless otherwise
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noted. The commercial bovine serum albumin digest was purchased from Michrom
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Bioresources.
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The E. coli digest was generated by hypotonic cell lysis followed by isolating the cytosolic
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fraction by centrifugation and subsequent digestion with trypsin at an enzyme to substrate
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ratio of 1:100.
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For the lapatinib selectivity assay, proteins were extracted from human placenta and kinases
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were purified in the presence and absence of 1 µM lapatinb (LC laboratories, MA) as
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described (Bantscheff et al Nat. Biotechnol, 2007).
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TMT labeling was performed as described (Bantscheff et al Nat. Biotechnol, 2007). 30 ng of
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TMT labeled BSA digest (TMT 126:127:128:129:130:131 = 1:2:4:8:16:32) was spiked into 6
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μg TMT-labeled E.coli digest (1:1:1:1:1:1).
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HPLC and Mass Spectrometry
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Mass spectrometry was performed on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher
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Scientific, Germany) coupled to a nanoLC Ultra 1D+ liquid chromatography system
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(Eksigent, CA). Peptides were separated using an in house packed trap column (20 mm x 75
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μm RepoSil-Pur C18, Dr. Maisch, Germany) in line with an analytical column (200 mm x 75
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μm RepoSil-Pur C18, Dr. Maisch, Germany) and gradient elution was performed from 2 to
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28% solvent B (0.1 M formic acid in 100% acetonitrile) within 4 h at a flowrate of 300 nl/min.
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The eluent was sprayed via emitter tips (PicoTip, New Objective, MA) using a nano-
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electrospray ion source (Proxeon Biosystems, DK). The mass spectrometer was operated in
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positive ion mode. Full scan MS spectra were acquired in the orbitrap recording a window
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between 300 and 1500 m/z at a resolution of 60,000 (at m/z 400) after accumulation to a
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target value of 500,000. CID of the five most intense ions was performed in the LTQ at a
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normalized collision energy of 35% after accumulation to a target value of 10,000 for max
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500 ms. Subsequently, HCD tandem mass spectra were triggered from the same five
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precursor ions. HCD ions were generated in the HCD collision cell using a normalized
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collision energy of 75%, a target value of 30,000 and 750 ms max accumulation time and
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subsequently detected in the orbitrap at 7500 resolution. Precursor ions were put on a
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dynamic exclusion list for 30 s. Internal calibration was enabled for both MS and MS/MS
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mode using the ion signal of (Si(CH3)2O)6 as a lock mass.
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Data processing
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The raw CID-HCD data was processed into Mascot searchable files (mgf) using Mascot
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Distiller (Matrix Science, UK). Two separate .mgf files were generated, each with peak
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processing and picking optimized for either peptide identification by CID or TMT
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quantification by HCD spectra. Briefly, uncentroiding of tandem MS spectra and isotope
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fitting was enabled for optimal CID scan processing (_CID.mgf). For HCD scans, peak lists
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were generated without further peak processing (_HCD.mgf). Then, the two .mgf files were
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merged into a single .mgf file using the provided Perl script (CID-HCDmerge). In short, for
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each HCD spectrum the TMT reporter ions were detected by their characteristic mass
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differences. The intensities of the reporter ions were extracted and their mass offset
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corrected. Further processing and visualization of the TMT intensities was done in MATLAB
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(The MathWorks, Germany). The processed reporter signals were pasted into the
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corresponding CID spectrum of the _CID.mgf file, deleting the respective m/z window at the
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same time (for more detail please see supplemental figure S1). Resulting peak lists
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(_CID_cut_HCD.mgf) were searched using the Mascot search engine version 2.3.01 (Matrix
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Science, UK). Data from the BSA/E.coli experiment was searched against the SwissProt
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database version 57.15 (515,203 entries). Data from the human samples were searched
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against the human International Protein Index (IPI) database version 3.78 (87,061 entries). A
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precursor tolerance of 10 ppm and a fragment tolerance of 0.6 Da was used in all cases.
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Enzyme specificity was set to trypsin and up to two missed cleavage sites were allowed.
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TMT 6plex at N-termini and lysine residues was set as fixed modification and, for the
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lapatinib selectivity data set, also carbamidomethylation at cystein. Variable modifications
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included N-terminal acetylation, oxidation of methionine, phosphorylation of serine, threonine
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and tyrosine and pyroglutamic acid formation of glutamine and glutamic acid, for the
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BSA/E.coli experiment in addition carboxy- (BSA) and carbamido- (E.coli) methylation at
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cystein. Quantification of TMT-labeled peptides was performed by Mascot without any further
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isotope corrections or normalization (already done within the pearl script).
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Other notes
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It should be noted that the pearl script we provide will only work for Mascot mgf files
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generated by Mascot Distiller from LTQ Orbitrap XL raw data files using a top1-5 CID
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followed by top1-5 HCD of the same precursors. Any other acquisition schemes and mgf
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processing will require modifications to the script (source code is provided). We provide the
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source code (file name: CID_HCD_Merge) and version compiled for Windows (file name:
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Win32_CID_HCD_Merge, which also contains a readme file describing how to run the
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script).
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Supplemental figures
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Figure S1: Configuration file of the provided script for processing data from combined CIDHCD acquisitions. Input data for the script are two .mgf files (_CID.mgf and _HCD.mgf), both
processed by Mascot distiller for the respective scan types. TMT6plex-labeled raw data must
be recorded in combined CID-HCD mode where up to five CID scans are followed by the
HCD scans from the same precursor. First the two corresponding spectra are identified by
their precursor mass within a window of 10 scans. TMT reporter ions are then detected in the
_HCD.mgf by their characteristic mass differences (theoretical mass differences calculated
from configured masses +/- tolerance distol) and their m/z values are corrected by the
median offset of all TMT signals within the spectrum. All identified peaks are extracted, their
intensities adjusted by the hcdfactor parameter and submitted to the Matlab software
(integrated in the merge script) for further peak processing to improve quantification. This
processing comprises isotope impurity correction of the isobaric tags by factors defined in the
configuration file, discarding of scans with missing reporter channels (tolmissvalfromleft;
most important for missing reference signals) and different normalization methods. Moreover
signal intensity distributions within their m/z value and against reference intensities are
visualized to estimate data quality. In a final step the refined reporter signals are merged in
the respective CID spectrum in the _CID.mgf where all peaks in a certain mass range around
the extracted ions (cidcuttol) are discarded. The resulting file format is again a .mgf file with
‘chimeric’ CID-HCD spectra for identification and quantification via Mascot. Further detail: the
top paragraphs relate to which fragment ions will be extracted from a given HCD spectrum. A
total of 9 TMT related ions are listed which include the six actual TMT channels as well as a
three related ions (125 for incomplete stable isotope labeling of the TMT126 reagent; 132
and 133 for the natural C13 isotopes of the TMT 131 reagent). In addition to the TMT ions,
any other ion of interest may be extracted from the HCD spectrum. Some immonium ions of
amino acids and the oxonium ion of HexNAc are shown as examples. The next section lists
the actual processing parameters (see above) which related to the mass tolerances with
which the difference processing and the HCD extraction and CID insertion are performed.
The script allows restricting processing up to a specified maximum LC retention time. The
remaining parameters relate to TMT quantification. Because the isotopic impurities of all TMT
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reagents, the relative contributions of one TMT channel to the others can be corrected. The
correction values are provided by the vendor and the script will optionally perform this step. It
is further possible to account for missing TMT channels and to normalize the data by two
different methods.
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Figure S2: TMT mass region extracted from data collected by LC.-MS/MS on a qTOF
instrument (ABSciex tripleTOF, see also figure 2 of the main manuscript). The data shows
that several additional ion signals are present in the TMT region but the presence of
interfering signals is a lot less pronounced than for the LTQ-Orbitrap XL. TMT ions are
detected with mass errors between 8 and 15 ppm.
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Figure S3: similar data as shown in figure 2A but when using two different batches of the
TMT reagents.
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Figure S4: Quantification distortion by the presence of near-isobaric signals. The example
spectrum (unprocessed data) shows the presence of one non-TMT ion near the TMT-128
channel and two such ions near the TMT-131 channel. When processing this data by Mascot
Distiller (our standard for processing raw Orbitrap files into Mascot searchable mgf files), The
TMT-128 intensity is erroneously increased. For the TMT-131 channel, both the processed
m/z value and the intensity are distorted. Mass difference processing of the raw data avoids
this distortion.
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Table S1: Possible chemical compositions calculated for the m/z species detected in the
TMT reporter ion region of the HCD data shown in figure 2.
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