High Throughput Screening in Drug Discovery for Cancer Research Peptides Using High
Resolution LCMS
Keeley Murphy1; Maciej Bromirski2; Francois A. Espourteille1 ;1 Thermo Fisher Scientific, San Jose , CA; 2 Thermo Fisher Scientific, Bremen, Germany
Peptide Name
Avg.
Std Dev
%CV
z-prime
Survivin 85-93
Samples were injected onto a C18, 42.1 x 30 mm, 3.5 µm HPLC column. Analyte elution
was accomplished using mobile phase A - water + 0.1% Formic Acid (v/v) and mobile
phase B - Acetonitrile + 0.1% Formic Acid (v/v) with a 1-minute step gradient at a flow rate
of 1.2 mL/min. The step gradient was performed at 0-10 sec, 2% B, 11-35 sec, 35%B, 3665 sec, 95% B, and 66-100 sec, 5% B. All methods were completed using a Thermo
Scientific Transcend LX-4 system with a dual injector arm with DLW (Dynamic Load and
Wash). LC multiplexing was implemented with a data window of 18 seconds per LC
channel.
Theoretical exact mass calculations were confirmed for each peptide by directly infusing
with 1 μg/mL solution of each compound at 5 μL/min. (Figure 2) The charge states
producing the most abundant signal for each peptide were determined during the infusion
procedure and the mass range for the full scan experiment set accordingly to monitor all
relevant charge states. Peptide charge states producing a low level response were
excluded from the data collection window to reduce overall data file size, with all charge
state ions below 10% of the total signal excluded from the collection range. Files
containing 96 or more injections were limited to an overall size of less than 500MB.
FIGURE 2. Top: Full scan peptide mix from 200 to 2000m/z. Bottom: zoomed scan
range from 200 to 950m/z.
Peptide_Infusion_10ug-mL
T: FTMS + p ESI Full ms [200.00-2200.00]
251.1608
100
466.2482
90
80
80
Missing Scans
52.63
60
52.52 52.55
52.59
52.61
52.61
52.60
Relative Abundance
30
52.63
52.57
52.55
800
Peptide_Infusion_10ug-mL
T:
FTMS + p ESI Full ms [200.00-2200.00]
251.1608
100
1000
1409.6700
1200
m/z
1527.7390
1400
1737.0157
1600
1800
2012.2716
2000
2200
52.65
Time (min)
Full Scan data collection window
52.71
52.73 52.74 52.76
52.70
1000
7.52874
0.380001
5.047339
100
0.62186
0.20801
33.44948
90
80
70
509.5845
613.6514
327.5356
272.1717
431.9845
597.8999
368.5936
0
300
400
500
657.9211
600
m/z
943.4185
763.8723
722.3664
700
766.9362
800
931.4894
900
0
368.2
368.4
368.8653
368.6
368.8
369.0
m/z
369.2198
369.2
369.4
1000
5.64208
2.483671
44.0205
30
368.7208
276.1339
369.6
369.9756
369.8
370.0
F:
31.8
32.0
32.2
FTMS + p ESI Full ms2 [email protected] [200.00-1000.00]
32.4
32.6
613.6508
100
623.2421
90
70
680.2655
571.3556
508.2194
466.7798
30
374.7191
20
354.2084
325.2048
200
736.4341
920.4725
779.3320
763.8685
832.9104
931.4531
911.4638
300
400
500
600
m/z
700
800
900
649.4022
533.3080
719.4057
849.5180
746.4187
300
594.3468
431.2617
589.3661
896.2707
0
200
40
0
453.2812
10
50
443.2971
294.1443
20
400
500
600
m/z
700
800
900
1000
FTMS + p ESI Full ms2 [email protected] [66.70-1000.00]
943.4174
100
Conclusion
Survivin 2B 80-88

Liquid chromatography with high resolution mass spec analysis provided a robust
and reproducible method for high throughput screening and simultaneous
analysis of multiple peptides at multiple charge states over the course of more
that 3000 individual sample injections.

Full scan data collection across a wide mass range enabled the acquisition of
spectral information for the entire isotopic charge envelope without sacrificing
data quality.

Statistical analysis of a large injection set resulted in Z’ values ranging from 0.61
to 0.85 indicating method robustness the analysis.

Multiplexing using a four-LC channel system provided rapid injection cycle times
of 30 seconds, on average, while proving separation from detergents and other
interferences containing in sample matrixes.

Full Scan / Full Scan AIF data collection allows for simultaneous Full Scan
quantitation as well MS/MS confirmation or quantitation with minimal reduction in
instrument scan speed.
624.2437
60
638.2805
50
607.2169
40
794.3123
693.2651
30
907.3964
711.2756
523.1965
812.3231
777.2831
20
535.2717
390.1436
306.1443
235.1074
10
506.1700
289.0960
889.3856
386.1664
881.3821
962.6227
0
300
400
500
600
m/z
700
800
900
1000
Bid BH3 +4
452.7581
588.3163
Bid BH3 85-98
60
631.8318
460.9855
40
40
502.2922
438.7608
461.2343
904.5093
705.4138
30
530.8033
20
388.7289
10
762.4361
948.4990
793.4769
677.4188
353.2104
228.1338
1003.5778
876.5143
339.2130
0
461.4847
459.9875
776.4509
50
200
300
400
500
600
m/z
700
800
900
1000
460.2342
460.2
460.4
460.6
460.8
461.0
m/z
461.2
461.4
4
FTMS + p ESI Full ms2 [email protected] [150.00-2000.00]
Conc (nM)
References
530.8096
100
90
508.2179
Bid BH3
5
10
100
1000
-- 0.00039 0.00097 0.01210 0.11674
18.11
16.75 16.72451 11.05993
z-prime
0.61
1. Zhang,J.-H.; Chung,T.D.Y.; Oldenburg,K.R. J. Biomol. Screening 1999, 4, 67-73.
70
2. Roddy,T.P.; Horvath,C.R.; Stout,S.J. Anal. Chem. 2007, 79, 8207-8213.
466.7800
50
40
20
344.8767
779.3337
431.2611
374.7192
580.3428
290.1454
709.8824
254.6124
0
200
300
AnaSpec is a trademark of AnaSpec, Inc. Gubbs is a trademark of Gubbs, Inc. Microsoft and Excel are registered
trademarks of Microsoft Corporation. All other trademarks are the property of Thermo Fisher Scientific and its
subsidiaries.
623.2443
354.2084
30
10
369.5966
31.6
10
550.3342
200
369.3950
368.3896
31.4
290.1460
422.2392
80
50
%CV
10
368.1898
10
257.1968
50
Bid BH3 Std Dev -- 7.1E-05 0.000163 0.002023 0.012911
30
460.4916
Survivin 85-93
60
60
20
Bid BH3
70
Avg.
369.1942
50
80
1
368.9938
NL: 5.57E6
m/z= 680.2622-680.2690 F: FTMS
+ p ESI Full ms2
[email protected]
[200.00-1000.00] MS
Curve_Plate_Run_7_253
Bid BH3 85-98
90
Peptide
Name
50
20
32.6
FTMS + p ESI Full ms2 [email protected] [66.70-1000.00]
233.1645
100
680.2656
534.3162
356.5467
32.4
80
30
40
32.2
0
60
60
0
50
Figure 5. Infusion of neat peptide solutions at 10uM conc. and resulting MSMS spectra
100
460.0
52.75
Bid BH3 +5
368.7933
40
50
32.0
Time (min)
Survivin 80-88
NL: 5.83E6
m/z= 631.8286-631.8350 F: FTMS
+ p ESI Full ms2
[email protected]
[200.00-1000.00] MS
Curve_Plate_Run_7_253
80
40
460.7391
60
60
31.8
Each peptide analyzed was interrogated for possible fragmentation prior to LC analysis.
Neat solutions of each peptide were individually infused to determine the peptide
fragmentation pattern. (Figure 5)
70
70
31.6
Survivin 85-93
NL: 6.34E5
m/z= 624.2406-624.2468 F: FTMS
+ p ESI Full ms2
[email protected]
[200.00-1000.00] MS
Curve_Plate_Run_7_253
Time (min)
F:
∆ppm = 2.7
80
80
31.4
368.5929
100
90
466.2482
**Interference
52.60
943.4185
1067.6248
600
100
0.78428
0.097311
12.4077
10
52.64 52.66 52.67
52.60
32.5
0
31.2
1000
1.28023
0.126222
9.859341
20
0
20
32.54
32.16 32.43
100
NL: 1.26E6
m/z= 422.2371-422.2413 F: FTMS
+ p ESI Full ms2
[email protected]
[200.00-1000.00] MS
Curve_Plate_Run_7_253
0
30
20
400
Bid BH3 +5
52.62
32.07
32.01
32.49
100
31.2
70
52.63
52.64
52.65 52.66
52.68 52.70 52.72
Relative Abundance
Ion source conditions were consistent for all MS experiment scan modes with the
following parameters held constant, Vaporizer temperature 500°C, capillary temperature
375°C, sheath gas 60, and AUX gas 20. Prior to experimental data collection, the
instrument was calibrated in positive ion mode using Pierce LTQ Velos ESI Positive Ion
Calibration Solution.
356.5467
200
31.73
31.63
31.53
32.43
FTMS + p ESI Full ms2 [email protected] [150.00-2000.00]
40
932.4922
32.06
31.69
31.28
0
50
90
20
534.3162
0
20
80
60
613.6514
32.02
31.22
31.23
7B
50
32.48
100
31.64
SM:
530.8094
90
40
50
90
Bid BH3 +4
80
10
100
0.133545
0.019558
14.64532
32.05
32.44
40
1000
12.96369
0.562784
4.34123
RT:
31.06 - 32.79
100
31.68
0
100
1.435063
0.16085
11.20856
T: FTMS + p ESI Full ms [350.00-615.00]
460.4938
100
52.62
100
60
40
31.65
31.18
FIGURE 3. Reanalysis of Bid BH3. (Top Left) Bid BH3 scans at +4 and +5, missing scans
indicate deviation outside of 5ppm analysis window. (Top Right) MS spectra at +4, ∆ppm =
7.1 indicate an unresolved interference. (Bottom Left) MS spectra at +5, ∆ppm = 2.7.
(Bottom Right) Table of recalculated Avg, Std. Dev., %CV, and z-prime.
0
100
Relative Abundance
A Thermo Scientific Exactive Plus bench-top Orbitrap mass spectrometer (Figure 1) was
used in Full Scan and Full Scan all-ions-fragmentation (AIF). Full Scan data was
collected across a mass range of 350-615 m/z at resolution 70,000 and a spectral speed
of 3 Hz. A maximum inject time of 250ms for the Full Scan was applied. Full Scan AIF
was collected with Full Scan across a mass range of 350-615 m/z at resolution 70,000
and a spectral speed of 3 Hz, and Full Scan AIF across a range of 200 – 2000 m/z at
resolution of 17,500 and a spectral speed of 12 Hz using a Normalized Collision Energy of
25. A maximum inject time of 250ms was applied for the Full Scan with 50ms applied for
the Full Scan AIF.
5
0.04581
0.007009
15.30206
31.22
32.05
32.02
FIGURE 6. MSMS extracted ion chromatograms (XIC) for all four peptide in simultaneous
analysis across a four channel multiplexing system. (Top) AIF XIC’s for each individual peptide
across a four channel system. (Bottom) AIF spectra at chromatographic peak apex.
32.46
32.45
32.47
31.66
31.65
70
70
Mass Spectrometry
5
0.02708
0.004446
16.41506
1
0.01718
0.003777
21.98924
Avg.
Std Dev
%CV
z-prime
Bid BH3
Relative Abundance
Liquid Chromatography and Multiplexing
Results
5
0.005237
0.000832
15.87972
1
0.00490
0.00094
19.18882
Avg.
Std Dev
%CV
z-prime
Bid BH3 85-98
5
0.063424
0.007772
12.25476
1
----
Avg.
Std Dev
%CV
z-prime
Survivin 2B 80-88
Sample Preparation
1
0.012532
0.001733
13.82914
Conc (nM)
10
0.135694
0.011968
8.820187
0.87
10
0.01118
0.001688
15.0943
0.71
10
0.05928
0.007849
13.24001
0.81
10
0.08430
0.01349
16.00155
0.32
32.03 32.04
31.66
31.21
31.19
60
Table 1. Mean, standard deviation, %CV and z-prime for each peptide analyzed. Highlighted
values indicate %CV and z-prime values above acceptable levels.
Methods
Five lyophilized peptides were ordered (catalog numbers: Survivin 2B (80-88) cat# 62693,
Survivin (85-93) cat# 62691, Bid-BH3 cat# 61631, Bid BH3 (85-98) cat# 62485 and
Substance P cat# 61694 from AnaSpec™, San Jose, CA). Each peptide was
reconstituted in a deionized water at 1 mg/mL. Serial dilutions of Survivin and Bid-BH3
were prepared at concentrations of 1 nM, 5 nM, 10nM, 100 nM, and 1000 nM in in a 50
mM HEPES solution containing 50 mM MgCl2, 50mM Citric Acid, Tween20, and 1% (v/v)
DMSO). Substance P was then added to each dilution level as an internal standard at a
final concentration of 100 nM.
80
31.20
Relative Abundance
FIGURE 1. Exactive Plus Instrument Schematic
0
100
Relative Abundance
Lead discovery groups are constantly developing new methodologies to screen large
chemical libraries against a specific target. The majority of these methods utilize plate
reader devices, ELISA, and other analytical techniques that are amenable to high
throughput analysis. Existing analytical methodology used for screening can be less than
ideal due to sample prep considerations, limit of detection, and specificity of analyte
detection. In these cases high resolution LCMS can provide a high quality alternative
while maintaining a sampling rate that is suitable for HTS requirements. Liquid
chromatography allows for analysis of samples in matrices that otherwise cause
interference in the MS source, while high resolution MS provides sensitivity and specificity
along with ease of use without compromising data quality. Two commercially available
peptide sets commonly targeted for cancer research were analyzed in a simulated
enzymatic screening assay experiment. Survivin 2B and Bid BH3 which are often
targeted for analysis for their role in cell apoptosis were monitored at various
concentrations in a high throughput screening experiment and the results analyzed to
determine signal response, linearity,-reproducibility, and Z’ value.
Thermo Scientific Pinpoint software was used for exact mass calculation of all peptides at
each of the observable charge states. Exact mass information was saved in Microsoft®
Excel® for later use. Thermo Scientific QuickCalc software (powered by Gubbs Inc., GMSU
Gubbs™ Mass Spec Utilities, Atlanta, GA) was used for all chromatographic data review,
and report generation. Compound information was imported into QuickCalc™ software
using an Excel import template containing the required m/z information. The compound
information was stored in QuickCalc software for later use. The compound information was
then grouped into a “compound set” and saved, allowing the exact m/z information for
multiple compounds to be easily recalled and applied to an entire data set for easy
chromatographic review. QuickCalc software was also used to automatically extract and
sort each of the 96 injections contained in a data file and then display individual
chromatographic peaks in a table view correlated with all sample information related to
each individual injection.
The fragmentation data collected during infusion was used to analyze AIF data collected during
Full Scan analysis. The most intense fragmentations for each peptide were used to generate
extracted ion chromatograms for the AIF data. The results demonstrate the presence of the
expected fragmentation patterns for all peptides with scan speed, peak shape, retention time, and
sensitivity adequate for MSMS confirmation as well accurate quantitation. The addition of the AIF
did not affect the quantitative results of the Full Scan component of the experiment due to the
rapid scanning speed of the MS.
FIGURE 4. AIF collection at 17,500 from 200-1000m/z. Scan settings provide for an average of
30 scans across the 6 second peak.
Relative Abundance
Introduction
Data Analysis Software
Relative Abundance
Results: Full scan mode at a resolving setting of 70,000 was utilized for the analysis of
several thousand sample sets at cycle time of 30 seconds per sample. Four individual
peptides were simultaneously analyzed with results demonstrating a linear signal
response across the working range of the assay with analyte sensitivity in the low nM
range. Full Scan operation at 70,000 resolving power enabled for data collection across a
wide mass range allowing for multiple peptide monitoring at multiple charge states and
detection of unexpected sample changes with no impact on instrument performance.
Relative Abundance
Methods: Several peptides commonly monitored for cancer research were analyzed at
various concentration levels in HEPES buffer solutions to determine signal response,
linearity,-reproducibility, and Z’ value.
Relative Abundance
Purpose: To implement high resolution accurate mass detection for the simultaneous
analysis of multiple components in a high throughput screening assay.
Sample sets were also collected using All Ion Fragment (AIF) to collect fragmentation
spectra for all peptides present in the sample injection. The Full Scan was collected at a
resolving power of 70,000 at 200m/z while the AIF was collected at a resolving power of
17,500 at 200m/z. The scans at the resolving power specified above allow for an average
of 30 scans across the six second wide chromatographic peak. (Figure 4)
Samples in HEPES Buffer solution were analyzed at 5 concentration points ranging
from 1nM to 1000nM with 288 replicate injections at each concentration level. The
average peak area ratio was used to calculate %CV at each concentration and
generate a calibration curve. (Table 1) The LOQ was determined to be 1nM for three
peptides and 5nM for the fourth, with a linear signal response across the detected
concentration range. Three of the peptides demonstrated a linear and reproducible
response with %CVs below 20% at all concentration levels. The fourth peptide,
although detectable at all concentration points displayed %CVs above 20% even at
increasing concentration levels. Z-prime statistical analysis provides an indication of
overall assay robustness based on the average and standard deviation of a positive
and negative control. Z-prime values above 0.5 indicate a robust and acceptable
assay. The z-prime for the forth peptide was well below 0.5 with a value of 0.32,
indicating a lack of robustness in the analysis. Upon subsequent examination an
unresolved interference was observed for the m/z of the fourth peptide that was not
present during assay method development. The m/z collection range utilized in the full
scan MS experiment allowed for reanalysis of the peptide at an alternate charge that
provided an acceptable but lower signal response. The average, standard deviation,
%CV, and z-prime was recalculated for the fourth peptide at the alternate charge state
through re-interrogation of the previously collect data. (Figure 3)
In all runs, multiple sample injections were collected into a single data file format using
Thermo Scientific Xcalibur and Aria software to automatically control data acquisition start
and stop times for each sample set. Commercially available peptides were analyzed at
various concentration levels and over multiple replicates to determine the coefficient of
variation, linear response, and lower limit of quantitation. Results for each experiment type
were compared for overall performance and ease of use.
Relative Abundance
Overview
400
500
600
m/z
700
789.3177
800
890.3651
932.5519
900
This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.
1000