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2
Experimental
Sample Preparation
K562 leukemia cells (ATCC) were grown in RPMI media
supplemented with 10% FBS. Cell lysates (1 mg) were
desalted using Thermo Scientific
™
7K Zeba
™
Spin Desalting
Columns and labeled with 5 µM of Thermo Scientific
ActivX
™
Desthiobiotin-ATP or -ADP probes for 10
minutes as described previously.
3
For inhibitor profiling,
cell lysates were pretreated with 0, 0.01, 0.3, 1, 3, and 10 µM
of staurosporine before addition of the desthiobiotin
nucleotide probes. Labeled proteins were reduced, alkylated,
desalted, and digested with trypsin. Desthiobiotin-labeled
peptides were captured using Thermo Scientific High-
Capacity Streptavidin Agarose Resin for two hours, washed
and eluted using 50% acetonitrile/0.4% TFA for MS analysis.
LC-MS
All separations were performed using a Thermo Scientific
EASY-nLC
™
nano-HPLC system and a binary solvent
system comprised of A) water containing 0.1% formic
acid and B) acetonitrile containing 0.1% formic acid. A
150 x 0.075 mm capillary column packed with Magic
™
C18 packing material was used with a 0.57% per minute
gradient (5%–45%) flowing at 300 nL/min at room
temperature. The samples were analyzed with a Thermo
Scientific Q Exactive
™
hybrid quadrupole-Orbitrap mass
spectrometer in either data-dependent or targeted fashion.
Details of the acquisition methods are summarized in
Tables 1 and 2.
Table 2. Mass spectrometer parameter settings used for targeted
experiments
Full
msxSIM
AGC Target Value
1 x 10
6
2 x 10
5
Max Injection Time (ms)
250
250
Resolution (FWHM at
m/z
200)
140,000
140,000
Isolation Window (Da)
500–1300
4
# Multiplexed Precursors
-
4
Data Analysis
Thermo Scientific Proteome Discoverer
™
software version
1.3 was used to search MS/MS spectra against the
International Protein Index (IPI) human database using
both SEQUEST
®
and Mascot
®
search engines.
Carbamidomethyl (57.02 Da) was used for cysteine
residue static modification. Desthiobiotin (196.12 Da)
modification and oxidation were used for lysine and
methionine residues, respectively. Database search results
were imported into Thermo Scientific Pinpoint
™
software
version 1.2 for high-resolution, accurate-mass (HR/AM)
MS-level quantitation. Data extraction was based on the
four most-abundant isotopes per targeted peptide. The
area under the curve (AUC) values were summed for the
total AUC values reported. The relative AUC values for
each of the isotopes were compared against the theoretical
isotopic distribution for further confirmation and
evaluation of potential background interference. The
half-maximal inhibitory concentration (IC
50
) values were
determined by plotting AUC values for each peptide
versus inhibitor concentration to generate a dose response
curve of inhibitor binding (K
d
) as described previously.
2
Results & Discussion
Building a Kinase Active-Site Peptide Library
While protein kinase sequences and active sites are readily
known from protein databases, it is still challenging to
build a method for detection, verification, and quantification
of kinase peptides. Our method focused on identifying
and quantifying kinase active-site peptides since a large
majority of these peptides are unique for their respective
kinase. In addition, these peptides provide direct insight
into kinase active-site inhibition for kinases that have
multiple kinase domains.
To build a list of kinase active-site peptides, untreated cell
lysate samples were labeled with the ActivX
Desthiobiotin-ATP or -ADP probes for active-site peptide
enrichment. An initial, unbiased Top10 data acquisition
method (Table 1) was used to generate spectral libraries
for database searching using Proteome Discoverer
software. These search results were used to determine
peptide sequences, desthiobiotin modification sites, and
protein kinase family members. In addition, this experiment
provided key data required for subsequent targeted
acquisition methods including peptide retention times,
precursor and product ion charge states, and HCD
product ion distribution.
Table 1. Mass spectrometer parameter settings used for discovery experiments
Parameter
Setting
Source
Nano-ESI
Capillary temperature (˚C)
250
S-lens RF voltage
50%
Source voltage (kV)
2
Full-MS parameters
Mass range (
m/z
)
380–1700
Resolution settings (FWHM at
m/z
200)
140,000
AGC Target
1 x 10
6
Max injection time (ms)
250
Dynamic Exclusion
™
duration (s)
70
Top n MS/MS
10
MS/MS parameters HCD
Resolution settings (FWHM at
m/z
200)
35,000
AGC Target
2 x 10
5
Max injection time (ms)
250
Isolation width (Da)
2
Intensity threshold
8 x 10
2
( 0.1% underfill)
Collision energy (NCE)
27
Charge state screening
Enabled
Charge state rejection
On: 1+ and unassigned rejected
Peptide match
On
Exclude isotopes
On
Lock mass enabled
No
Lowest
m/z
acquired
10