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