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Effective coupling of CE with nanoESI MS via a true sheathless metal-coated emitter interface for robust and
high sensitivity sample quantification
Keqi Tang, Xuejiang Guo, Thomas L. Fillmore, Yuqian Gao
ASMS 2016
Biological Sciences Division, Pacific Northwest National Laboratory
Characteristics of capillary electrophoresis mass spectrometry (CE-MS)
♦ High separation efficiency.
♦ Broad analyte coverage including structure variants.
♦ Sensitive detection (an intrinsic low flow separation technique).
♦ Easy to set up.
Limitations
♦ Small sample loading capacity.
♦ Lack of a robust CE-MS interface.BGE BGE
Separation Capillary
CE or CZE separation, Background electrolyte (BGE): 9:1 volume ratio of 0.1 M acetic acid in deionized water to methanol.
♦ Combined sample stacking (focusing) and separation to allow a sample loading volume up to 1/3 of the total separation capillary volume (~ 2 to 3 uL).
♦ Selective enrichment of low abundance species in a complex mixture.
♦ Mobility dependent focusing:
A effective solution to limited sample loading capacity
Trailing electrolyte (TE): 9:1 volume ratio of 0.1 M acetic acid in deionized water to methanol.
Leading electrolyte (LE): 25 mMammonium acetate in deionized water with the solution pHadjusted by adding acetic acid to pH = 4.
A {[µL (µL + µR )] [(µA + µR ) µA]}.C = LC
Sample Loading
Sample Stacking
Separation
Sample in LE
Transient capillary isotachophoresis (CITP/CZE):
♦ Electric contact for ESI through conductive liquid enclosed in a short metal tube and porous wall of the emitter capillary.
♦ Separation capillary: 100 µm i.d., 360 µm i.d., 95 cm long, 7.5 µl total volume for large sample loading; etched ESI emitter capillary: 20 µm i.d., 90 µm o.d. for stable NanoESI.
♦ No sample dilution and easy exchange of emitter capillary.
♦ Limit of quantitation: 25 attomoles ( at 60 nL/min ESI flow rate).
Two previous interfaces for online CITP/CZE-SRM MS
ESI voltage
High Voltage Power Supply
TE
Syringe
Gas
CITP/CZE ESI
Separation capillary ESI emitter capillary
a) Sheath liquid interface*:
b) Sheathless interface**:
♦ Electrical contact for ESI through the liquid junction at the emitter tip.
♦ ESI voltage at the waste line to avoid gas bubbles for stable electrospray operation.
♦ Separation capillary: 75 µm i.d., 150 µm i.d., 85 cm long, 4 µl total volume; laser pulled ESI emitter capillary: 200 µm i.d., 360 µm o.d., ~50 µm tip.
♦ Limit of quantitation: 50 attomoles (at ~100 nL/min ESI flow rate).
* C. Wang et al., Anal. Chem., 84, 10395-10403 (2012); ** C. Wang et al., Anal. Chem., 85, 7308−7315 (2013).
Weakness of the interfaces: ♦ Require careful adjustment and control of fabrication process to
achieve reproducible and sensitive sample analysis.
♦ Difficult to operate for an inexperienced user.
Development of a robust sheathless CE-MS interface*
♦ A commercial CE capillary (360 µm o.d., 30 µm i.d.; 100 cm long) with an integrated metal coated ESI emitter having a constant i.d. and tapered o.d..
♦ Electric contact for ESI through conductive liquid enclosed in a short metal tube and the metal coated outer surface of the emitter.
Pressured gas
CE voltage power supply
Conductive liquid
SRM MS
Tapered ESI emitter with metal coated outer surface
BGE
CITP/CZE
Movable Teflon sleeve
Metal tube filled with conductive liquid
ESI voltage power supply
* X. Guo et al., Anal. Chem., 88, 4418−4425 (2016).
ESI flow rate: 5.7 nL/min; Sample: a mixture of 9 peptides at 2.0 µM each in leading electrolyte; Labeled peaks from 1 to 9 are Kemptide, Substance P, Bradykinin, Angiotensin I, Angiotensinogen (1-14), Neurotensin, Angiotensin II, Leucine Enkephalin, Fibrinopeptide A, respectively.
Effect of sample injection volume on CITP/CZE-MS with the new sheathless CITP/CZE-MS interface
Rel
ativ
e Ab
unda
nce
(%)
Migration time (min)
100
18 20 22 24 26 28 30 32 34 36 38 40 420
50
NL: 8.95E7227.2 nL (32.2%)
100
0
50
NL: 8.16E7198.8 nL (28.1%)
20 22 24 26 28 30 32 34 36 38 40 4218
0
50
100 NL: 7.11E7170.4 nL (24.1%)
20 22 24 26 28 30 32 34 36 38 40 4218
0
50
100 NL: 4.05E7113.6 nL (16.1%)
20 22 24 26 28 30 32 34 36 38 40 4218
0
50
100 NL: 2.27E756.8 nL (8.0%)
20 22 24 26 28 30 32 34 36 38 40 4218
0
50
100 NL: 1.92E728.4 nL (4.0%)
20 22 24 26 28 30 32 34 36 38 40 4218
1
2 38
9
76
54
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
7
7
7
7
7
8
9
89
8 9
8 9
8 9
Effect of sample injection volume on peak area
0.0E+00
5.0E+08
1.0E+09
1.5E+09
2.0E+09
2.5E+09
3.0E+09
0 50 100 150 200 250
Peak
are
a
Sample volume (nL)
Angiotensin IAngiotensin IIBradykininFibrinopeptide AKemptideLeucine EnkephalinNeurotensinAngiotensinogen (1-14)Substance P
ESI flow rate: 5.7 nL/min; CE separation voltage: 30 kV; ESI voltage: 2.2 kV.
Effect of ESI flow rate on analyte peak migration time and peak intensity
05
1015202530354045
2.5 7.5 12.5 17.5
Mig
ratio
n tim
e (m
in)
Flow rate (nL/min)
0.0E+0
5.0E+6
1.0E+7
1.5E+7
2.0E+7
2.5E+7
2.5 7.5 12.5 17.5
Pea
k in
tens
ity
Flow rate (nL/min)
Angiotensin I
Angiotensin II
Bradykinin
Fibrinopeptide A
Kemptide
Leucine Enkephalin
Neurotensin
Angiotensinogen (1-14)
Substance P
Sample loading volume: 28.4 nL; CE separation voltage: 30 kV; ESI voltage: 2.2 kV.
Effect of separation voltage on analyte peak width and total separation window
0
0.1
0.2
0.3
0.4
0.5
17.5 22.5 27.5
Peak
FW
HM
(min
)
Separation voltage (kV)
Angiotensin I
Angiotensin II
Bradykinin
Fibrinopeptide A
Kemptide
Leucine Enkephalin
Neurotensin
Angiotensinogen (1-14)
Substance P0
5
10
15
20
17.5 22.5 27.5
Sep
arat
ion
win
dow
(min
)
Separation voltage (kV)
Sample loading volume: 28.4 nL; ESI flow rate: 5.7 nL/min; ESI voltage: 2.2 kV.
CITP/CZE-MS analyses of 50 nM BSA tryptic digest at different sample injection volumes
ESI flow rate: 5.7 nL/min; ESI voltage: 2.2 kV; CE voltage: 30 kV.
Migration Time (min)
22 24 26 28 30 32 34 36 38 40 42 44 460
20
40
60
80
100
Rela
tive
Abun
danc
e (%
)
0
20
40
60
80
100582.56572.11
449.83653.62 464.44
941.10
740.53708.68
874.67
432.96515.00
408.94
477.17747.97
653.60
464.53
582.51740.81
474.19
461.99
487.88756.80572.12449.83
722.70708.70941.13 554.45642.58432.99 752.13748.09
NL: 2.18E8, Base Peak MS
NL: 3.32E7, Base Peak MS
198.8 nL (28.1%)Peak capacity: ~90
28.4 nL (4.0%)Peak capacity: ~130
CITP/CZE-SRM MS quantitation of Kemptide
Targeted peptides (spiked in 50 nM BSA digest matrix) and monitored transitionsCompound Name Sequence Precursor Ion (m/z) Product Ion (m/z)
Kemptide [LRRASLG+2H]2+ 386.7 567.3 (b5+-NH3), 409.3 (b3
+-NH3), 539.4 (a5+-NH3)
y = 0.0244x - 0.0009R² = 0.9927
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 20 40 60 80 100 120
Peak
are
a (×
108 )
Sample amount (picomole)
0
0.5
1
1.5
2
2.5
0 0.5 1 1.5Pe
ak a
rea
(×10
6 )
Sample amount (picomole)
5 amol
Migration Time (min)22 23 24
0
1000
2000
3000
10 amol
Migration Time (min)22 23 24
0
2000
4000
6000
8000
ESI flow rate: 5.7 nL/min, ESI voltage: 2.2 kV, CE voltage: 30 kV, Sample loading volume: 198.8 nL.
Sensitivity, reproducibility and linearity of the CITP/CZE-SRM MS quantification of Kemptide
Concentration (nmol/L) Amount (amol)
Average migration time (min)
CV of migration
time Average area CV of area0.025 5 23.1 0.7% 614x103 25.2%0.05 10 22.5 1.3% 2.14x104 5.0%0.1 20 23.3 0.6% 4.35x104 24.6%0.5 99 22.8 1.8% 2.02x105 20.3%5 994 24.7 0.4% 1.93x106 12.9%
50 9.94x103 23.7 0.5% 2.55x107 12.3%100 1.99x104 23.0 0.2% 4.71x107 9.2%250 4.97x104 22.7 0.6% 1.21x108 5.2%500 9.94x104 23.3 0.7% 2.42x108 9.5%
♦ Robust new sheathless CE-MS interface: the stability of system remained after more than a hundred sample analyses without the noticeable loss of the metal coating on the emitter surface.
♦ The new sheathless CITP/CZE-MS interface design allows robust and reproducible CE-MS analysis.
♦ The new interface allows stable and dilution free nanoESI operation at the flow rate as low as 5.7 nL/min, improving both CITP/CZE separation quality and MS detection sensitivity.
♦ The durability of the new sheathless CITP/CZE-MS interface was verified by over a hundred sample analyses without the noticeable loss of the metal coating on the emitter surface and the clogging of the emitter.
♦ High sensitivity CITP/CZE-NanoESI SRM MS quantification of targeted peptides in BSA digest matrix was demonstrated with LOQ as low as 5 attomoles.
Conclusions:
Funding Support:♦ NIH National Cancer Institute for Biomarker Verification♦ NIH National Cancer Institute for Glycan Characterization♦ NIH National Institute of General Medical Sciences
Acknowledgements
Other members of CITP/CZE-SRM MS development team:
Chenchen Wang Ronald J. MooreTujin Shi Ryan Kelly