Comprehensive Profiling of the Proteome,
Lipidome, and Metabolome Enabled Using a
Prototype UPLC-Compatible Microfluidic Device
J. Will Thompson 1, Jay Johnson2, Giuseppe Astarita2, Giuseppe
Paglia2, Jim Murphy2, Steven Cohen2,
Jim Langridge4, Geoff Gerhardt2, and M. Arthur Moseley1
1Duke
Proteomics Core Facility, Durham , NC;
2Waters
3Center
Corporation, Milford, MA;
for Systems Biology, University of Iceland,
4Waters
Corporation, Manchester, UK
Benefits and Compromises of Changing Column
Diameters for Various Applications
0.075 mm
Benefits
Compromises
2-4x Speed
& Efficiency/time
4-5x sample
required
1/20 Solvent/Sample
Consumption
10-20% increase in
time
20-40x Sensitivity
Source/ionization
flexibility
Benefits
Compromises
0.150 mm
2.1 mm
Must be weighed for each individual application
Tile Design and Flow Diagram
ESI Emitter
Assembly
Incoming flow
Electrical Connections
(EEPROM, Heater)
Analytical Column
Trap Column
Evaluation Areas for Prototype 150 um Tile
Label-Free Quantitation, Proteomics
ug EColi, 5 fxn, 37 min grad, HDMSE, Fxn 5
UCA195
23:05:17
D09049_01_UCA195_3252_092712_05
22.85
995.21
100
%
8.94
478.81
7.62
585.84
13.28
622.88
1: TOF MS ES+
BPI
1.31e5
22.91
995.21
39.16
795.79
38.46
32.42
987.55
39.26
964.08 34.83
1022.21
750.96
43.99
995.54
0
5.00
10.00
D09049_01_UCA195_3252_092712_04
15.00
12.57
10.76;720.95 549.84
100
9.26
613.40
20.00
25.00
18.19
858.47 18.34
858.47 20.63
15.63
983.53
724.43
30.00
35.00
40.00
1: TOF MS ES+
BPI
9.02e4
24.96
995.54
7.11
617.37
%
Targeted Peptide Quant,
Method Development and Deployment
30.30
28.48
706.75
1196.27
20.31
748.48
18.32
748.92
27-Sep-2012
28.75
774.41
31.62
748.42
4.43;409.25
32.74
935.25
39.16
949.15
0
5.00
10.00
D09049_01_UCA195_3252_092712_03
15.00
16.25
901.51
100
%
7.11
8.79 10.00 13.53
510.81 613.35 602.00 586.36
6.17;486.33
25.00
30.00
35.00
5.00
10.00
D09049_01_UCA195_3252_092712_02
100
6.64
4.20 878.98
562.64
9.94
859.96
1: TOF MS ES+
BPI
1.27e5
18.69
777.36
15.00
12.23
645.02
14.45
575.32
40.00
16.35
901.51
26.14
812.92
21.54 23.03
792.97 798.98
34.98
1257.62
29.63
832.42
Metabolomics (RPLC and HILIC)
0
%
20.00
20.00
25.00
30.00
38.44
1310.63
35.00
Lipid Profiling (Flow Injection)
40.00
1: TOF MS ES+
BPI
7.68e4
17.62
888.78
20.67
926.77
3.47
548.32
26.82
739.70
24.40
533.02
26.93
739.70
32.21
1133.05
39.19
1003.55
43.91
765.08
0
5.00
10.00
D09049_01_UCA195_3252_092712_01
%
100
15.00
20.00
25.00
30.00
35.00
3.43 3.47;811.01
811.01
3.22
590.32
8.90
6.52 517.28
642.87
18.87
870.42
40.00
39.19
1003.55
16.07
14.26 526.28 17.27
590.83
655.30
10.59
573.83
ID09969_01_UCA195_3302_030513.raw : 1
23.57
995.22
22.99
652.42
23.91
1014.23
29.32
1196.29
34.96
30.28;836.72 750.98
1: TOF MS ES+
BPI
6.61e4
39.26
1022.22
0
Time
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
ID09969_01_UCA195_3302_030513.raw:1
Summary of Multi-Omics Sample Preparation Strategy
Cell Disruption
(Sonication in AmBic pH8)
Bradford Assay, 1.8mg/sample
(normalize by total lysate)
Polar Metabolites
~48%
80/20 MeOH/water
1 hr extraction, N2 dry
Lipids
~48%
80/20 MTBE/MeOH
1 hr extraction, N2 dry
Proteins
~4%
0.25% w/v Rapigest
DTT/IAA/trypsin overnight
Resuspend
2/1/0.2 MeCN/
Formic Acid/HFBA
Inject 1% for LC-MS/MS
(30 min/sample)
Resuspend
4/2/1
IPA/MeOH/CHCl3
Inject 4% for FIA
(10 min/sample)
Acidify
1/2/97 TFA/MeCN/water
Inject 20% for 2DLC-MS/MS
(3 hr/sample)
150 um Prototype Tile
Direct Inject/Flow Injection Fluidic Diagram
Sample
Needle
2
1
3
6
4
A
150
5
B
Analytical Pump
um Tile
Tile options tested:
5, 10, 20 cm
BEH C18
HSS T3 C18
CSH C18
BEH C4
BEH Amide HILIC
Infusion Tile
RPLC Metabolomics Method
Arginine
m/z
BPI
MP B Compos.
Analysis used 1% of isolate:
150 um x 10 cm 1.7 um BEH C18 tile, F = 2.0 ul/min at 45°C
Mobile phase A: 0.1% Formic acid, 0.02% HFBA, in water
Mobile Phase B: 0.1% Formic acid in 10/90 IPA/MeCN
Mass Spectrometry: Synapt G2 HDMS, Resolution mode (25,000 Rs) @ 5Hz
>17,000 metabolite
Features in 12 minutes
Phenylalanine
S-adenosyl methionine (SAM)
5-methylthioadenosine (MTA)
Time (min)
Lipid Profiling using Flow Injection
Analysis and an Infusion Tile
Analysis of the Lipid Isolate from MCF7
cells (prepared using MTBE/MeOH
extraction).
- Ion-Mobility Data-Independent Analysis
- Synapt G2, 0.6 sec scans (6V or 15-45V)
- 3 ul/min flow rate
- Mobile phase was 10/90 IPA/MeCN
with 0.1% formic acid
m/z
ID09969_01_UCA195_3302_030513.raw : 1
Ion Mobility
Approximately 600 unique lipid species quantified in a 4 minute run (5 min cycle)
ID09969_01_UCA195_3302_030513.raw:1
150 um Prototype Tile
2D with Dilution Fluidics
RP1
- Xbridge-BEH130 C18 NanoEase Column, 5μm, 300 μm x 50 mm
Trap
- UPLC Symmetry C18 Trap, 5 μm, 180 μm x 20 mm
MS/MS - Synapt G2 – hdDIA (hdMSE)
Goals for High-Throughput Proteomics
Analysis Using 2DLC and TRIZAIC
Time per sample (hr)
0
1
2
3
4
Type
Column
F
F
/min
1D
Nano*
112
0.8
2D
Nano*
TriZAIC
295*
405
1.0
1.35
2D
TriZAIC
350
2.0
2D**
TriZAIC
350
2.6**
5
*
90 min gradient @ 0.4 ul/min
*
37 min gradient @ 0.4 ul/min (nano) or 3 uL/min (Tile)
18.5 min gradient @ 3 uL/min (Tile)
**
18.5 min gradient @ 3 uL/min (Tile)
Initial Trapping Step
Fraction Elution to 2nd Dimension
Analytical Separation
* Current “standard” configurations
**Potential elimination of between-fraction trapping time
with dual-trap 2DLC prototype (K. Fadgen and M. Staples)
2D LC/MS/MS on Synapt G2
nanoLC vs 150 um Tile
75 um x 150 mm BEH C18 column
7 to 35% MeCN in 37 min, 0.5 ul/min
20:24:34
100
16.52
956.68
18.57
657.76 21.03 23.55
551.85 550.35
29.61
995.29
27.23;748.53
25-Sep-2012
29.79
995.29
37.15
34.63
706.81
760.01
39.24
964.16
%
14.86
691.98
39.73
649.40
12.99;604.41
0
5.00
10.00
ID09141_02_UCA195_3241_092512_04
100
15.00
20.00
25.00
30.00
35.00
40.00
1: TOF MS ES+
BPI
1.29e5
26.21
26.29;858.53
858.53
31.45
28.12 995.61
983.59
15.20 16.66 20.20
601.90 785.50 550.38
14.86
617.91
12.62;550.86
%
1: TOF MS ES+
BPI
42.97
988.13 1.43e5
37.11
774.98
42.86
39.81 1033.30
935.57
02:47:38
ID09156_01_UCA195_3252_092712_05
100
13.09
995.54
6.21
730.43
100
28-Sep-2012
1: TOF MS ES+
BPI
1.28e5
16.99 20.47
706.75 987.55
20.56
927.15
5.50
585.84
0
5.00
10.00
15.00
ID09156_01_UCA195_3252_092712_04
39.24
1122.20
9.32;443.30
7.07;720.94
5.90;648.84
4.18
550.80
10.84
858.46
20.00
25.00
30.00
35.00
40.00
1: TOF MS ES+
BPI
9.77e4
25.00
30.00
35.00
40.00
1: TOF MS ES+
BPI
1.54e5
25.00
30.00
35.00
40.00
1: TOF MS ES+
BPI
1.04e5
30.00
35.00
40.00
1: TOF MS ES+
BPI
7.02e4
30.00
35.00
40.00
1: TOF MS ES+
BPI
39.16
1.31e5
14.09
17.89
995.53
935.49
20.51;1118.03
0
0
20.00
25.00
23.75
902.07
100
%
18.24
15.82
14.06 613.39 822.51
11.74 486.35
9.32 716.50
449.33
0
5.00
10.00
ID09141_02_UCA195_3241_092512_02
100
14.20
11.05
879.02
562.67
15.00
20.14
834.97
20.00
35.00
40.00
1: TOF MS ES+
BPI
1.87e5
23.85
902.07
24.20;901.55
25.00
34.32
812.96
34.59
29.67
792.52 31.47 812.96 38.16
832.47
964.10
30.00
20.20
645.05
24.59
21.33 902.04
28.50
682.40
926.82
17.79
860.50
9.80
601.42
%
30.00
28.59
926.82
8.17
453.28
35.00
42.78
1257.70
41.13
1208.32
40.02
35.94
1133.11
761.75
9.84
901.99
7.79
645.01
14.94
812.90
20.41
1310.85
5.00
10.00
15.00
ID09156_01_UCA195_3252_092712_02
100
7.83
3.53 645.34
562.30
20.00
8.15
682.35 11.99
926.75 15.10
774.69
22.83
765.05
0
10.80
590.35
9.65
749.46
10.86
590.35
15.00
14.69
643.39
9.02;494.30
20.00
25.00
30.00
35.00
40.00
1: TOF MS ES+
BPI
1.21e5
17.99
517.30
18.44
517.30
23.34 25.94
590.85 526.30
27.83 31.48
870.45 995.26
34.38
784.83
7.95
409.24
38.73
1196.34
0
42.89
751.00
Time
10.00
15.00
20.00
25.00
30.00
35.00
40.00
5.00
10.00
15.00
ID09156_01_UCA195_3252_092712_01
3.53
100
738.88
20.00
25.00
4.88;642.86 9.86
526.27 13.44
995.20
20.47
1003.53 22.89
525.93
%
5.00
10.00
ID09141_02_UCA195_3241_092512_01
5.00
5.31
510.80
43.25
345.12
0
100
100
20.00
0
40.00
1: TOF MS ES+
BPI
1.33e5
34.73
739.74
5.00
10.00
15.00
ID09156_01_UCA195_3252_092712_03
%
15.00
%
5.00
10.00
ID09141_02_UCA195_3241_092512_03
%
3 ug EColi, 5 fxn, 18p5 min grad, HDMSE, Fxn 1
%
UCA195
ID09141_02_UCA195_3241_092512_05
%
HDMSE, 3 ug
150 um x 100 mm BEH C18 nanoTile
7 to 35% MeCN in 18.5 min, 3.0 ul/min
0
5.00
10.00
15.00
ID09049_01_UCA195_3252_092712_05
100
8.94
18.32
20.00
22.85
995.21
25.00
22.91
995.21
30.30
706.75 32.42
795.79
TRIZAIC 150 2DLC Configuration
Chromatographic Evaluation versus 75 um Capillary Column technology
Acknowledgments
Duke University Proteomics Core Facility
http://www.genome.duke.edu/cores/proteomics/
Funding
NIH S10 grant
Duke School of Medicine
CTSA grant UL1RR024128