A Novel 6 × 5 Peptide Mixture for Instrument Performance Monitoring
Michael Rosenblatt, Ethan Strauss, and Marjeta Urh
Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711
Introduction
Monitoring LC and MS Parameters
Chromatography and Mass Spec
(A)
Hydrophobicity
VTSGSTSTSR
Total Ion Intensity (TIC)
(A)
LASVSVSR
VVGGLVALR
YVYVADVAAK
LLSLGAGEFK
LGFTDLFSK
509.3(m/z)
428.2(m/z)
459.8(m/z)
566.8(m/z)
1X [Pep]
537.3(m/z)
535.3(m/z)
90
85
0.1X [Pep]
530.3 (m/z)
504.7 (m/z)
424.2 (m/z)
562.8 (m/z)
456.3 (m/z)
502.2 (m/z)
420.7 (m/z)
559.8 (m/z)
453.3 (m/z)
499.7 (m/z)
417.7 (m/z)
496.7 (m/z)
75
532.3 (m/z)
450.3 (m/z)
525.3 (m/z)
447.3 (m/z)
553.8 (m/z)
0.01X [Pep]
525.3 (m/z)
0.001X [Pep]
521.8 (m/z)
528.8 (m/z)
0.0001X [Pep]
518.3 (m/z)
521.8 (m/z)
21.15
65
60
55
50
LGFTDLFSK
45
38.17
40
30
25
20
15
10
5.73
5
504.7
562.8
420.7
502.2
499.7
532.3
(Mass/charge)
530.3
(Mass/charge)
504.7
(B)
420.7
502.2
417.7
499.7
(C)
562.8
459.8
(D)
456.3
537.3
(E)
532.3
453.3
559.8
556.8
553.8
414.7
496.7
566.8
535.3
(F)
530.3
525.3
528.8
450.3
525.3
521.8
521.8
447.3
10
518.3
y = 1.0754x + 6.2629
R² = 0.9854
9
8
Log (mole)
Log (fmole)
Log (fmole)
500 amol
5
50 amol
+
Retention Time (Minutes)
Peptides elute in
correct order
+
Total Ion Current
+
Peptides have good
linear fit
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]
Retention Time
100
559.82
95
566.83
553.81
90
5
4
0
2
Log (fmole)
4
-2
9
7
y = 1.0007x + 6.196
R² = 0.9811
8
7
6
y = 0.8401x + 6.5318
R² = 0.9894
5
4
3
4
-2
0
2
Log (fmole)
0
2
Log (fmole)
65
4
11
10
9
8
7
6
5
4
3
y = 0.8023x + 6.3033
R² = 0.9664
-2
0
2
Log (fmole)
55
Total Ion Intensity (TIC)
554.31
50
45
40
35
30
25
20
554.81
15
557.31
560.32
563.33
567.33
YVYVADVAAK
8/23
8/25
8/27
8/29
8/31
9/2
Date of Measurement
9/4
9/6
9/8
9/10
LASVSVSR
LGFTDLFSK
1.E+08
LLSLGAGEFK
1.E+06
VVGGLVALR
1.E+04
YVYVADVAAK
1.E+02
VTSGSTSTSR
8/23
8/25
8/27
8/29
8/31
9/2
Date of Measurement
9/4
9/6
9/8
•
30 peptides: 6 sequences × 5 isomers per sequence
•
10-fold abundance gradient from “heaviest” isomer to “lightest”
•
Complementary software provided to report LC and MS parameters
as well as tracking historical performance or comparing instruments
•
Mixture can be analyzed neat or spiked into complex samples
•
All peptides are analyzed by AAA quantified so as to insure
accurate reporting of sensitivity and dynamic range
9/10
557.81
10
559.32
560.82
(Time(min) )
556.31
563.83
561.32
561.82
558.81
558.32
555.31
553.31
566.33
562.32
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
4
Conclusions
VVGGLVALR
In addition to having individual LC and MS parameter reports, the software tool will also analyze
parameters like Peak Height and Retention Time. Additional parameters like mass accuracy, linear fit,
lowest detectable mass etc., can also be analyzed.
60
4
LGFTDLFSK
LLSLGAGEFK
8
0
2
Log (fmole)
y = 0.9471x + 6.202
R² = 0.9672
5
3
-2
10
5
6
4
9
6
7
VTSGSTSTSR
70
Relative Abundance
y = 0.8903x + 6.406
R² = 0.9874
6
8
LLSLGAGEFK
75
P1>P2>P3 etc.
7
LGFTDLFSK
80
5
8
556.81
85
Pep 1
Pep 2
Pep 3
Pep 4
Pep 5
9
LASVSVSR
1.E+10
1.E+00
8/21
562.82
45
Panel A. Representative chromatogram. Panel B. A plot of the log Peak Intensity versus log (molar
amount) provides a measure of the instruments Dynamic Range and confirmation of the instrument
sensitivity. Optimal loading conditions are first determined by titration to identify the region of optimal
instrument linearity.
Peak Height (Total Ion Current)
Heaviest Peptide has
sufficient intensity
Log (intensity)
Instrument Parameter History
1.E+12
Peptides
coelute
44.08
YVYVADVAAK
9
10
-2
Scoring Function:
40
3
0
2
Log (fmole)
3
25
22.5
20
17.5
15
12.5
10
7.5
5
8/21
35
4
4
Reanalyze
with mass
accuracy
adjustments
42.46
30
10
VVGGLVALR
Increase
initial
mz tolerance
and reanalyze
• Select best peaks
25
Time (min)
10
3
Log (fmole)
Panel A. Chromatographically, 6 peptides elute. However, each peptide is a mixture of 5 isotopologues
which can be seen in an XIC analysis as shown in the figure and which will be available in the software.
Panel B. This feature is clearly seen in the MS spectra of the eluting peak. Panel C. A plot of the log of
MS intensity versus the log of the molar amount on the column. The mixture is able to confirm up to 5 logs
of dynamic range per run and is used to confirm the lowest detectable amounts per run. Assuming proper
titration initially, this can also be used to confirm LOQ and LOD.
For each peptide sequence
500 fmol
5 fmol
6
-2
Algorithm Workflow:
20
30.17
LASVSVSR
50 fmol
7
4
Log (mole)
15
34.48
26.39
21.65
18.16
VTSGSTSTSR
(Mass/charge)
Log (intensity)
Log (Peak Height)
(A)
428.2
424.2
10
15.44
518.3
(B)
509.3
13.08
521.8
521.8
(Mass/charge)
5
10.34
525.3
525.3
447.3
8.39
535.3
(F)
528.8
450.3
553.8
(Mass/charge)
537.3
(E)
453.3
556.8
414.7
(Mass/charge)
456.3
559.8
417.7
496.7
459.8
(D)
Log (intensity)
424.2
566.8
(C)
Log (intensity)
428.2
(B)
Log (intensiy)
Log (intensity)
(B)
509.3
Log (intensity)
(A)
Log (mole)
Reanalyze with time constraints if
peptides elute in the wrong order
29.39
YVYVADVAAK
35
Software Analysis
• Identify times at which multiple
isotopologues co-elute
LLSLGAGEFK
24.91
12.59
70
556.8 (m/z)
414.7 (m/z)
VVGGLVALR
LASVSVSR
80
(Time(min) )
(C)
• Identify all peaks for each
isotopologue with the correct isotopic
signature
NL:
1.11E9
Base Peak F:
FTMS + p NSI
Full ms
[375.001600.00] MS
msb16749_04
95
Relative Abundance
Performance monitoring of LC-MS/MS instrumentation continues to
be challenging and has yet to be standardized across all MS
laboratories. Towards this end, we have prepared a peptide
mixture; in combination with an accompanying software tool, this
mixture reports on LC column performance and MS instrument
parameters, including sensitivity and dynamic range. In addition to
reporting on all critical instrument parameters, the software can
report on parameter history, compare instruments, and display XIC
traces for the peptides. This is the first example of a peptide
mixture designed to give a reporting of all critical LC and MS
parameters in a single run.
VTSGSTSTSR
RT: 1.72 - 45.08
3.84
100
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