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Optimizing Capillary LC/MS PeptideOptimizing Capillary LC/MS PeptideOptimizing Capillary LC/MS PeptideOptimizing Capillary LC/MS PeptideAnalysisAnalysisAnalysisAnalysis
Jeffrey L. Holyoke and Steven A. Cohen
Waters CorporationMilford MA
030525
Experimental Design and System
• Compare and Contrast Sensitivity andMobile Phase effects on UV and MSDetection.
• Examine Methods for High ThroughputCapillary LC/Tandem MS Methods
• System– Waters CapLC, Waters LCZ 4000 or
Micromass QTOF030526
Tryptic Map Overlays (n = 10)
AU
0.00
0.20
0.40
0.60
Minutes
10.00 20.00 30.00 40.00 50.00 60.00 70.00
Conditions:Sample: 16 pmols on column Bovine Cytochrome cA 0.1% TFAB 0.085% TFA in MeCNColumn: Symmetry Packed 0.32x150 mm 100Å, 5←←←← particle size3% B to 43% B in 80 minutes at 5 ←←←←L/minMean Standard Deviation for Retention Time < 0.15
030527
Evolution of Capillary Detector Design
Photodiode
Photodiode
Photodiode
Transverse Illumination- short pathlength- low sensitivity
Shaped Cell- long pathlength- better sensitivity- stray light- poor linearity
Light Guided- long pathlength- good linearity- near quantitative throughput
Light source
Light source
Light source Flow In
Flow OutFiber Optics
Pat #5,184,192
030528
How Capillary LC IncreasesSensitivity
Increase in concentration =(Dlarger)
2
or(Dsmaller)
2
(3.9)2
(0.32)2 = 149 times
Smaller diameter = lower peak volume
Lower peak volume = higher concentration
030529
Comparison of Capillary toStandard Chromatography
Columns: Waters Symmetry 100™ C18, 100Å, 5← particle size
0.00
0.12
AU 1 2 3
4 5 6
7 8
9
10
11
1213
20 60Minutes
0.00
0.20
AU1
2 34
5 6
78
9
10
11
12 13
Alliance System (analytical HPLC)3.9 mm ID Column
F = 0.8 mL/min810 pmol Sample
CapLC™ System0.32 mm ID Column
F = 0.005 mL/min20 pmol Sample
030530
320 micron vs 180 micronUV at 214 nm detection of 8 pmols
50.0Time
100
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0-0
%
1.64e541.7136.33
3.23 31.56
29.7926.6823.14
7.73 24.23 33.91
39.8637.51
38.78
46.88
44.54
0.180 x 150 mm Symmetry C18, 100A, 5u2 uL/min, 0.01% TFA
-0
100
%
1.64e529.45
18.4516.8215.6314.222.32
5.92
24.6524.47 27.02
28.03
31.6534.00
0.320 x 150 mm Symmetry C18, 100A, 5u5 uL/min, 0.01% TFA
030531
6.00 10.00 14.00 18.00 22.00 26.00 30.00 34.00 38.00Time6
100
%
6
100
%
1: Scan ES+ TIC
3.32e5
1: Scan ES+TIC
3.32e5
Comparison of TIC8pmol Injected
1 32
4 56
9
8 13
1211
10
7
1312
10
11
9/8
76
542/31
0.1% TFA
0.01% TFA
030532
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z0
100
%
0
100
%
1: Scan ES+ 8.38e3817
545617
618
818
1: Scan ES+8.38e3
817
545
617616
818
Comparison of Spectral QualityPeak 10, 8 Pmol
0.1% TFA
0.01% TFA
030533
Comparison of UV Response 8 Pmol, UV at 214 nm
6.00 10.00 14.00 18.00 22.00 26.00 30.00 34.00 38.00Time
0
0
100
%
100
%
2149.00e4
2149.00e4
1 3
2 4 5
6
9
8
13
1211
10
7
13
12
10
11
9/8
7
6
54
2/3
1
0.1% TFA
0.01% TFA
030534
High Sensitivity MS Detection with0.01% TFA
10.00 20.00 30.000
100
%
0
100
%
1005816585
728
588 792634
1005817
585
728633
589 792
TIC 300 - 2200 M/Z60 micron ESI probe16 vs. 1.6 pmol
030535
0.05% Acetic Acid vs. 0.05% Acetic Acid + 0.005% HFBA, UV at 214 nm
5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00
2: Diode Array 214
1.05e5
2: Diode Array 214
1.05e5
Time-1
100
%
-1
100
%
12
10
11
8/9
7
6
3
2
1
5
13
1
2/34
5
6 98
13
11
10/12
7
4
030536
0
100%
0
100%
m/z = 589 6.99e3Area
3491
m/z = 589 4.13e3Area
2537
Acetic Acid
Acetic Acid + HFBA
GNVEK
0
100%
0
100%
1634
1787
m/z =729 3.60e3Area
m/z = 729 4.04e3Area
Time0
100%
0
100%
576
1200
Acetic Acid
Acetic Acid + HFBA
GITWGEETLMEYLENPK
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
m/z =10051.48e3Area
m/z = 10053.35e3Area
TGQAPGFSYTDANK Acetic Acid
Acetic Acid + HFBA
Variable Ion Suppression with Acetic Acid and Heptafluorobutyric Acid
030537
LC/MS/MS Objectives
• Produce a fully integrated analytical system– From sample submission to protein identity
• Identify proteins in an automated fashion
• Increase Sample throughput (>6 samples /Hr)
030538
LC/MS/MS Conditions
• Sample :ß -Galactosidase trypticdigest
• 180µmx150mm PepMap C18• Flow rate 2µL/min• A = 0.1% Formic Acid• B = 0.1% Formic Acid in MeCN• Qtof: Data dependent MS\MS mode
Gradient I (post-loading): 5 - 60%B in35 min
Gradient II (Stream Select): High flowload at 5 % B for 3.2 minutes, 5 - 60%linear in 1 minute with a hold for 3.5minutes and subsequent return toinitial conditions
030539
030540
Nanospray LC/MS Peptide MapGradient I
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00Tim e0
100
%
0
100
%
0
100
%
0
100
%
19.1716.33
14.92
13.7913.37
12.60
18.88
17.65
19.4622.77
22.40
20.5025.80
23.3428.20
19.4216.63
15.25
13.9113.59
12.62
19.17
18.09
17.42
20.2122.61
21.8621.35 26.0422.79
23.33 25.40
26.44
28.46
19.2816.62
15.33
14.14
13.90
12.82
19.06
17.94
17.25
20.1122.44
21.37
24.7322.83
25.70 26.19
19.3816.66
15.02
13.99
13.71
12.71
19.09
18.21
17.34
20.31
22.45
21.4924.7122.74 25.11
26.14
CapLC- Q-Tof ß -Galactosidase tryptic digest
BPI
BPI
BPI
BPI
125fmoles injected
125fmoles injected
250fmoles injected
250fmoles injected
Flow rate 2µL/min5 - 60%B in 35 min0.1% FA/MeCN
030541
100fmole injection B-galactosidase trypticdigest Gradient II
100fmole injection B-100fmole injection B-galactosidasegalactosidase tryptic trypticdigest Gradient IIdigest Gradient II
6.04
4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 Time0
100
%
5.89
5.635.535.324.86
5.07
6.55
6.18
7.226.75
Base peak intensity (BPI) chromatogramBase peak intensity (BPI) Base peak intensity (BPI) chromatogram chromatogram
030542
4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 Time0
100
%
4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 Time0
100
%
6.04
5.89
5.635.535.32
4.865.07
6.55
6.18
7.226.75
Selected m/zchromatograms
Desired MW = 681.43at Retention time = 6.55
030543
tryptic fragment T24 res 300-310tryptic fragment T24 res 300-310tryptic fragment T24 res 300-310
100 fmole ϒ-galactosidase 100 fmole 100 fmole ϒϒ-galactosidase-galactosidase
m/z 681.4 (2+)m/z 681.4 (2+)m/z 681.4 (2+)
666 668 670 672 674 676 678 680 682 684 686 688m/z0
100
%
681.43
681.92
682.44
682.94
030544
666 668 670 672 674 676 678 680 682 684 686 688m/z0
100
%
681.43
681.92
682.44
682.94
MS modeMS modeMS mode
Preset intensity thresholdPreset intensity thresholdPreset intensity threshold
Automatic MS/MSAutomatic MS/MSAutomatic MS/MS
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z0
100
%
662.38
141.11
86.09
300.17
272.17
187.09
270.12 387.20301.17 587.27440.22
542.26
1062.56
663.38
775.45
664.35
904.49776.43
777.48
975.52
976.51
1063.55
1064.55
030545
Summary
• Capillary HPLC is becoming a routine analysis tool• New instrument design can provide sub-picomole
UV and MS detection• Direct flow can give reproducible gradients in the
low microliter per minute range• LC/MS/MS analysis now capable of providing
femtomole level sequence analysis
030546
Thanks to Everyone
• Waters– Dennis DellaRovere– Joe Luongo– Frank Rubino– John Heden– Scott McLaren
• Micromass– Bob Bordoli– Chris Hughes
• Glaxo-Welcome– Walter Blackstock– Jyoti Choudhary– Malcolm Ward
• MaxEnt Solutions– John Skilling
030547
