Mike Rosenblatt, Ph.D.
The 6 x 5 LC-MS/MS Peptide
Reference Mixture and
Software Analysis Tool (PReMiS™ )
October 2014
Promega Corporation
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Reference Materials for Mass Spectrometry
•
System Suitability and Quality Control
•
•
Evaluation of Sample Prep Methods
•
•
Sensitivity, Dynamic Range, Separation, etc.
Method Development: Proteolysis, Enrichment, Fractionation, etc.
Instrument Method Development
•
Optimization of Instrument Parameters for Discovery Experiments
•
Optimization for Quantitative Experiments (SILAC, iTRAQ, SRM)
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2
A Peptide Mixture for LC-MS/MS Methods
Development and QC
•
Mass Spec (MS) instruments are utilized in research, drug
discovery, disease monitoring, drug testing, environmental
and food safety, etc.
•
These instruments need to be monitored to avoid sacrificing
precious samples.
•
No reagent exists to determine all LC and MS parameters, in
particular sensitivity and dynamic range in a single
experimental run.
•
What if we could introduce a single reagent to report all key
instrument performance parameters in a single run?
•
And what if we provide (free) software to make it even
easier?
Promega Corporation
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3
Designing an Idealized Peptide Mixture
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Peptide Selection Process
Large collection of Peptides from Human Serum
List reduced to 8282 Unique Peptides
Removed P,M,W,C,N,Q, N-terminal E/D
Kept peptides between 7 and 15 AA long
Rejected insoluble and synthetically difficult
Synthesized 96 crude peptides
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5
Retention Time Analysis of 96 Crude Peptides
45
Retention Time (min.)
40
35
30
Bin 1
Bin 2
Bin 3
Bin 4
Bin 5
Bin 6
25
20
15
10
5
0
12 14 8 4 17 25 23 26 27 35 58 59 49 56 60 75 77 69 65 7 76 84 94 83
Peptide Identifier
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6
Final Selection
Large collection of Peptides from Human Serum
List was reduce to 8282 Unique Peptides
Removed P,M,W,C,N,Q, N-terminal E/D
Kept all peptides between 7 and 15 AA long
Rejected insoluble and synthetically difficult
Synthesized 96 crude peptides
Narrowed list down to 28 final candidates
Decided on final, six peptides
Promega Corporation
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7
(Total Ion Current)
MS Peak Intensity
A mixture of 6 Peptides…or is it?
most hydrophilic peptide
C18 LC Gradient (increasing hydrophobicity)
most hydrophobic peptide
LC Chromatogram
Peptide Retention
Time (minutes)
LASVSVSR
VVGGLVALR
LGFTDLFSK
VTSGSTSTSR
YVYVADVAAK
LLSLGAGEFK
Producing a set of peptides for LC monitoring is straightforward…
But how will we measure MS sensitivity and dynamic range?
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8
LC Gradient (increasing hydrophobicity)
(Total Ion Current)
MS Peak Intensity
Sensitivity and Dynamic Range in one LCMS/MS run
LC Chromatogram
Peptide 1
Peptide 6
(most hydrophobic)
(most hydrophilic)
Peptide Retention Time (minutes)
Intensity
Mixture Five isotopologues
of Peptide 1
* * **
* * *
**
*
*
*
*
*
*
*
*
*
*
*
Heavy
Labeled
AA’s
•
Sensitivity and dynamic range analysis accomplished
via incorporation of stable isotopes
•
Mixture of 5 isotopologues (differ only by mass)
•
Each mixed in different amounts to look at sensitivity and
dynamic range…
(mass)
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9
Dynamic Range in One Experiment
Sequence
MW
DM
1
LLSLGAGEFK
1072.67318
0.00
2
LLSLGAGEFK
1062.64598
10.03
3
LLSLGAGEFK
1055.62878
7.02
4
LLSLGAGEFK
1048.61158
7.02
5
LLSLGAGEFK
1041.59448
7.02
Intensity (log scale)
1 pmole
0.1
pmole
0.01
pmole
0.001
pmole
0.0001
pmole
Peptide Mass (m/z)
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Corrected Intensity
Isomer #
1 pmole
0.1 pmole
0.01 pmole
Linear
Dynamic
range
0.001 pmole
0.0001 pmole
Lowest
Detected
amount
Peptide Mass (m/z)
10
A mixture of 6 x 5 = 30 peptides for complete
monitoring of LC-MS/MS parameters
Hydrophobicity
Total Ion Intensity (TIC)
(A)
VTSGSTSTSR
LASVSVSR
509.3(m/z)
VVGGLVALR
YVYVADVAAK
428.2(m/z)
LLSLGAGEFK
459.8(m/z)
566.8(m/z)
LGFTDLFSK
1X [Pep]
537.3(m/z)
535.3(m/z)
0.1X [Pep]
530.3 (m/z)
504.7 (m/z)
424.2 (m/z)
562.8 (m/z)
456.3 (m/z)
532.3 (m/z)
502.2 (m/z)
420.7 (m/z)
559.8 (m/z)
453.3 (m/z)
528.8 (m/z)
499.7 (m/z)
417.7 (m/z)
556.8 (m/z)
450.3 (m/z)
525.3 (m/z)
496.7 (m/z)
414.7 (m/z)
553.8 (m/z)
447.3 (m/z)
521.8 (m/z)
0.01X [Pep]
525.3 (m/z)
0.001X [Pep]
521.8 (m/z)
0.0001X [Pep]
518.3 (m/z)
(Time(min) )
(C)
Log (Peak Height)
(B)
Log (intensity)
(A)
509.3
428.2
(B)
424.2
504.7
(Mass/charge)
509.3
(A)
504.7
(Mass/charge)
428.2
424.2
420.7
502.2
417.7
499.7
496.7
Log (mole)
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(Mass/charge)
566.8
562.8
(C)
Log (mole)
537.3
(E)
532.3
559.8
556.8
459.8
456.3
453.3
450.3
Log (mole)
521.8
518.3
(Mass/charge)
537.3
(E)
532.3
Log (fmole)
(Mass/charge)
535.3
530.3
(F)
525.3
528.8
525.3
521.8
447.3
530.3
525.3
525.3
521.8
(Mass/charge)
(D)
535.3
(F)
528.8
450.3
447.3
553.8
414.7
456.3
453.3
556.8
553.8
(B)
459.8
(D)
559.8
417.7
414.7
496.7
562.8
420.7
502.2
499.7
566.8
(C)
Log (fmole)
521.8
518.3
Log (fmole)
11
Peptide Components
Residues in red correspond
to amino acids in which all
carbon atoms are 13C and
all nitrogen atoms are 15N.
This is how we impart the
mass difference.
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12
Characterization and Quality Testing of the
6 x 5 Peptide Mixture
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Chromatography
RT: 1.72 - 45.08
3.84
100
95
Conditions:
Buffer A: 0.1 % Formic Acid
Buffer B: 0.1 % Acetonitrile
Gradient: 0 – 40 % B in one hour
Column: 0.075mm x 15 cm C18 column
NL:
1.11E9
Base Peak F:
FTMS + p NSI
Full ms
[375.001600.00] MS
msb16749_04
VTSGSTSTSR
90
VVGGLVALR
LASVSVSR
85
80
24.91
12.59
29.39
YVYVADVAAK
75
70
LLSLGAGEFK
21.15
65
60
55
LGFTDLFSK
50
45
38.17
40
35
30
25
20
15
10
5
5.73
8.39 10.34
5
10
13.08 15.44 18.16
15
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20
21.65
25
Time (min)
26.39
34.48
30.17
30
42.46 44.08
35
40
45
14
Isotope Spacing
3.5 Da
3.5 Da
3.5 Da
Peptide
MW
LLSLGAGEFK
LLSLGAGEFK
LLSLGAGEFK
LLSLGAGEFK
LLSLGAGEFK
1041.594
1048.612
1055.629
1062.646
1072.673
Z=2
52
52
52
53
53
5 Da
This spectrum, where peptides are mixed 1:1:1:1:1, exemplifies a typical mass spectrum. Because each mass is at
least 5 daltons (Z=1), we see no “crosstalk” between isotopic envelopes.
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15
In Process Check: AAA correlates with MS intensity
Because we perform AAA we are able to normalize all concentrations per isotopologue.
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16
16
Establishing Full Dynamic Range, LOD and LOQ
LASVSVSR (Heavy isotope; 428.27)
10
13.61
50
5.05 5.91 8.50 9.76 11.56
0
100
NL: 3.20E9
m/z= 427.77-428.77 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
MSB15830_1pmol082213
14.07 15.40
13.63
20.27 22.71 24.42 25.49
14.09 17.07
13.57
20.28 22.74 24.43 25.54
NL: 2.14E9
m/z= 427.77-428.77 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
msb15830_500fmol082213
500 fmol
50
5.07 6.39
0
100
9.20 11.52
NL: 4.95E8
m/z= 427.77-428.77 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
msb15830_100fmol082213
100 fmol
50
5.06 5.90
0
100
10.14 11.52
50 fmol
50
5.85
0
100
8.39
11.49
13.76 15.42
13.53
14.08 16.34
13.58
5.10 6.87 8.13 10.72
0
100
1 fmol
50
5.78
0
100
14.08 16.40
13.60
16.41
5.06
0
100
NL: 3.33E7
m/z= 427.77-428.77 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
msb15830_10fmol082213
19.96
22.67 24.42 24.98
20.04
24.48 25.08
26.42
24.77
22.68
16.36
7.20 9.21 11.05
19.96
24.97
25.32
13.51
100 amol
50
5.19
0
0
5
22.68 24.96
9.02 11.25 12.68
10
16.32
15
Time (min)
0.1 fmol
1 fmol
-1.5
-0.5
0.5
1.5
2.5
3.5
Log (fmole)
19.92 22.67 24.39 25.59
500 amol
50
7
NL: 2.32E8
m/z= 427.77-428.77 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
msb15830_50fmol082213
22.69
8.67 10.23 13.04
13.52
1 pmol
8
19.99 22.72 24.61 26.06
10 fmol
50
9
6
20.21
20
25.99
NL: 3.06E6
m/z= 427.77-428.77 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
msb15830_1fmol082213
NL: 2.15E6
m/z= 427.77-428.77 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
msb15830_500amol082213
NL: 1.84E6
m/z= 427.77-428.77 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
msb15830_100amol082213
Log (Peak Height)
1 pmole
100
Log (Peak Height)
RT: 0.00 - 29.01
10
9.5
9
8.5
8
7.5
7
6.5
6
y = 1.0695x + 6.4962
R² = 0.9975
0
0.5
1
1.5
2
2.5
3
Log (fmole)
25
This should be done first to establish the optimal loading condition.
In this example, 0.5 – 1 pmol would be optimal.
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17
Experimental Determination of Instrument
Dynamic Range
500 fmol
50 fmol
5 fmol
500 amol
50 amol
-2
0
2
10
9
8
7
6
5
4
3
4
y = 0.8903x + 6.406
R² = 0.9874
-2
Log (fmol)
0
2
Log (fmol)
4
Log (intensity)
Log (intensity)
4
y = 0.9471x + 6.202
R² = 0.9672
-2
0
2
4
Log (fmol)
LGFTDLFSK
LLSLGAGEFK
y = 1.0007x + 6.196
R² = 0.9811
-2
2
10
9
8
7
6
5
4
3
Log (fmol)
VVGGLVALR
10
9
8
7
6
5
4
3
0
Log (intensity)
y = 1.0754x + 6.2629
R² = 0.9854
YVYVADVAAK
11
10
9
8
7
6
5
4
3
y = 0.8401x + 6.5318
R² = 0.9894
Log (intensity)
10
9
8
7
6
5
4
3
LASVSVSR
Log (intensiy)
Log (intensity)
VTSGSTSTSR
9
7
y = 0.8023x + 6.3033
R² = 0.9664
5
3
-2
0
2
Log (fmol)
4
-2
0
2
4
Log (fmol)
Instrument: Thermo Q-Exactive. A dynamic range of 5 orders is confirmed as well as a minimum sensitivity of 50 amol.
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18
Multi-Lot Comparison
Real Time Stabilty by MS Analysis
Peptide 2
14
12
12
10
Pilot 2
8
First Batch (R and D)
6
4
y = 1.07x + 9.6908
R² = 0.9895
0
-5.00
-4.00
-3.00
-2.00
-1.00
Linear (First Batch (R
and D))
14
12
10
Pilot 2
8
First Batch (R and D)
6
4
y = 0.9243x + 9.0497
R² = 0.9958
2
0
-5.00
0.00
-4.00
Log (pmole)
-3.00
12
10
Pilot 2
8
First Batch (R and D)
6
4
y = 0.9409x + 9.588
R² = 0.9939
-3.00
-2.00
-1.00
Linear (First Batch (R
and D))
0.00
Log (pmole)
Log (Intensity)
Log (Intensity)
14
12
-4.00
Pilot 2
8
First Batch (R and D)
6
4
y = 0.9242x + 9.0232
R² = 0.9926
2
0
-5.00
0.00
-4.00
-3.00
-1.00
0.00
Peptide 6
14
12
10
Pilot 2
8
First Batch (R and D)
6
4
y = 0.9762x + 9.6627
R² = 0.9967
2
0
-5.00
-2.00
Linear (First Batch (R
and D))
Log (pmole)
Peptide 5
14
0
-5.00
-1.00
10
Log (pmole)
Peptide 4
2
-2.00
Linear (First Batch (R
and D))
-4.00
-3.00
-2.00
Log (pmole)
-1.00
0.00
Linear (First Batch (R
and D))
Log (Intensity)
2
Peptide 3
Log (Intensity)
14
Log (Intensity)
Log (Intensity)
Peptide 1
10
Pilot 2
8
First Batch (R and D)
6
4
y = 1.008x + 9.6123
R² = 0.9932
2
0
-5.00
-4.00
-3.00
-2.00
-1.00
Linear (First Batch (R
and D))
0.00
Log (pmole)
Stability after 1.5 years in real time at –20°C (stored in solution)
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19
Data Analysis: Time Consuming and Tedious
But, there is a solution!
A PC Based Software tool:
• Optional LC-MS/MS full configuration setup – for documenting
purposes
• LC and MS single analysis report
• XIC Analysis
• Analysis of Linear Fit
• Analysis of Parameter History
• Multianalysis Report
• Compatible with .raw, wiff, and .mzml formats
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20
Internal Scoring Function enables confident peak
Detection (especially complex samples)
Peptides
coelute
Score:
+
Heaviest peptide
have sufficient
intensity
+
Peptides have
good linear fit
Peptides elute in
Correct Order
+
Heaviest Peptide
has correct
isotopic pattern
+
MSB16751_04 #3874-4154 RT: 20.50-21.53 AV: 101 NL: 1.12E8
T: FTMS + p NSI Full ms [375.00-1600.00]
562.82
100
559.82
95
566.83
553.81
90
556.81
85
80
75
70
Total Ion Intensity (TIC)
65
Relative Abundance
Pep 1
Pep 2
Pep 3
Pep 4
Pep 5
60
55
554.31
50
45
40
35
30
557.31
560.32
563.33
567.33
25
20
557.81
10
P1>P2>P3 etc.
5
(Time(min) )
554.81
15
556.31
562.32
560.82
563.83
561.32
561.82
558.81
558.32
555.31
553.31
559.32
566.33
567.83
564.33
0
553
554
555
556
557
558
559
560
561
m/z
562
563
564
565.83
565.32
565
566
568.33
567
568
569.81
569
570
Scoring allows selection of correct peptide, especially in complex backgrounds.
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21
PReMiS™
Software Tool for
Peptide Data Analysis
Promega Corporation
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Software Front End
Promega Corporation
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23
Processing A New Sample
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24
Reporting LC and MS Parameters
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25
Reporting on Chromatography of all Peptides
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26
Instrument Linearity and Sensitivity
Confirms good sensitivity in linear range (1 fmol)
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27
Tracking Parameter History …
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28
Comparing Analysis Across Instruments…
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29
Documenting Instrument Setup
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30
Applications
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Finding Peptides in Neat Samples is Easy
RT: 0.00 - 57.00
3.84
100
12.59
21.15
24.91
29.39
38.17
50
3.50 5.73 8.39
3.84
100
50
0.77
0
100
*
5.04 8.26
13.08 16.79
34.48
25.69
55.28
42.46 45.15 48.46
NL: 1.49E9
m/z= 508.76-509.76 F: FTMS + p
NSI Full ms [375.00-1600.00]
MS MSB16749_04
14.03
12.59
3.71 6.04 8.47
0
100
19.82 22.13
29.42
34.20 37.27
44.81 46.94
53.15 56.26
NL: 8.53E8
Base Peak m/z= 427.76-428.76
F: FTMS + p NSI Full ms
[375.00-1600.00] MS
MSB16749_04
16.56 18.58
21.15
26.23 29.28
36.21 38.26
43.86
50.45
56.03
NL: 7.43E8
Base Peak m/z= 566.32-567.32
F: FTMS + p NSI Full ms
[375.00-1600.00] MS
MSB16749_04
*
50
3.84
0
100
8.12 12.85 17.25 20.17
22.82
24.91
3.96 5.64 8.26
12.66
16.90
29.26 31.11
24.05
3.84 5.31
12.68
26.13 29.40 33.63 38.28
29.39
18.40 21.19
28.37
*
30.17
3.88
0
5
7.59
12.65 14.92
10
15
21.18 24.90
20
25
Promega Corporation
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29.39
30
Time (min)
44.41 46.95
55.97
NL: 8.31E8
Base Peak m/z= 536.84-537.84
F: FTMS + p NSI Full ms
[375.00-1600.00] MS
MSB16749_04
38.13
38.17
50
0
51.18 56.50
NL: 8.61E8
Base Peak m/z= 459.31-460.31
F: FTMS + p NSI Full ms
[375.00-1600.00] MS
MSB16749_04
50
0
100
38.13 40.02 43.67
*
50
0
100
* Software determined RT
54.82
*
50
NL: 1.11E9
Base Peak F: FTMS + p NSI Full
ms [375.00-1600.00] MS
MSB16749_04
44.94
53.40 55.54
NL: 4.65E8
Base Peak m/z= 534.82-535.82
F: FTMS + p NSI Full ms
[375.00-1600.00] MS
MSB16749_04
*
36.80
39.90 42.67 48.89 52.09 56.29
35
40
45
50
55
32
Harder for Complex Samples…
unless you have software
RT: 0.00 - 116.00
NL: 1.60E10
TIC MS
Promega_K562_15830_1pmol
96.69
100
50
10.55
50
85.37 92.79
101.45
111.36
NL: 2.39E8
m/z= 508.77-509.77 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
Promega_K562_15830_1pmol
51.66
*
100
82.61
63.19
51.05 62.19
26.74 33.76
11.92 19.57
54.22
6.34
28.13
15.40 26.74
0
100
38.47
34.70
64.45 70.44
73.22
47.03
100.95
86.08
89.52
101.62
*
NL: 1.69E9
m/z= 427.76-428.76 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
Promega_K562_15830_1pmol
22.55
50
6.37 9.34
0
100
21.83
24.62
35.76
46.69
43.86
37.90
50
6.34 9.77
0
100
24.90
55.07
64.68 69.66
81.23
89.60
NL: 1.02E9
m/z= 566.32-567.32 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
Promega_K562_15830_1pmol
57.80 62.85
52.04
72.91
81.85 83.51
103.23
113.72
NL: 1.44E9
m/z= 459.30-460.30 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
Promega_K562_15830_1pmol
*
50
8.00 12.74 21.33 26.74 34.70
0
100
60.61
43.50
65.40 76.45
65.32
87.87 92.71 99.46 112.80
NL: 1.33E9
m/z= 536.84-537.84 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
Promega_K562_15830_1pmol
*
50
10.69 18.67 23.73
0
100
26.59
35.70 50.86 57.96
74.47 81.72 86.24 98.08 99.83
83.20
*
50
12.87 14.33 15.73 33.66 39.36 45.87
0
0
10
20
30
40
Promega Corporation
©2013 Promega Corporation. Proprietary Information. Not for further distribution.
* Software determined RT
106.01 114.21
*
26.92
Peptides were spiked
(1 pmol) into Human
K562 digest
56.69 61.56 64.90 76.75
50
60
Time (min)
70
80
85.33
90
100.61
100
NL: 1.18E9
m/z= 534.80-535.80 F: FTMS + p
NSI Full ms [300.00-1600.00] MS
Promega_K562_15830_1pmol
113.22
110
33
Comparing Instruments
Linearity (r2)
Retention Time
25.00
1.05
20.00
1.00
15.00
0.95
OVP
10.00
QE1
5.00
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QE2
0.80
Signal-to-Noise most abundant peptide
5000
4000
3000
2000
1000
0
QE1
0.85
QE2
0.00
OVP
0.90
Peak Width
OVP
QE1
QE2
50
40
30
20
10
0
QE1
QE2
FTIT
34
Can we Assess Instrument Precision?
25.00
20.00
15.00
10.00
5.00
0.00
S/N (MAP) Variance
2000.00
Signal/Noise
Time (minutes)
Retention Time Variance
1500.00
1000.00
500.00
0.00
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
S/N (LAP) Variance
Signal/Noise
Slope (Log:Log plot)
Linear Analysis Variance
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
Analyzing the mixture multiple times, along with software analysis, is a great way to assess instrument
precision.
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35
Summary
o 30 peptides: 6 sequences x 5 isomers per sequence.
o 10-fold abundance gradient from “heaviest” isomer to “lightest”.
o One injection gives up to 5 logs of dynamic range and can track
sensitivity down to the attomole level
o Complementary software provided to report LC and MS parameters
as well as tracking historical performance or comparing instruments
o Software can read .raw (Thermo), .wiff (AB Sciex) , and .mzml
formats
o Mixture can be analyzed neat or spiked into complex samples
o All peptides are analyzed by AAA quantified so as to insure accurate
reporting of sensitivity and dynamic range
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36
Comparing the Peptide Mix with Human/Yeast
Extracts
Task/Application
Product
Frequency
Rationale
Routine Instrument
QC
6 x 5 LC-MS/MS
Peptide Reference
Mixture
1-4X per day
(or spiked into
every sample)
The 6 x 5 mix gives confirmation that
the instrument is working. Customers
will want to know this as frequently
as possible
Method Development
Digested Human or
Yeast Extracts with
Peptides spiked in
Weekly –
especially after
column or
hardware
changes
• Gives a record of instrument
performance and will give some
indication for the frequency of
maintenance and calibration
• Optimization of complex mixture
analysis
• Targeted quantitation
Sample Prep
Workflows
Undigested Human
and Yeast Extracts
1-2X per
month
depending on
needs for
workflow
development
• Optimization of proteolysis
• Fractionation optimization
• Enrichment optimization
Promega Corporation
©2013 Promega Corporation. Proprietary Information. Not for further distribution.
37
Product Format
Amounts
(pmol)
Volume
(µL)
[Peptide]
(pmol/µL)
Estimated
# of
injections
Liquid
25
50
0.5
50
$125
Up to 10µL/min
Lyophilized
200
NA
NA
40-100
$550
Greater than
10µL/min
Format
Price
HPLC Flow Rate
• Two formats will be delivered to accommodate large customer base
• Formats were chosen so as to maximize resale value
• Software is complimentary to any user of the product
Promega Corporation
©2013 Promega Corporation. Proprietary Information. Not for further distribution.
38