Enhancing Sensitivity and Peak Capacity with Sheathless CE-ESI-MS
1
Busnel ,
2
Schoenmaker ,
2
Ramautar ,
2
Carrasco-Pancorbo ,
Jean-Marc
Bart
Rawi
Alegria
Jeff D. Chapman1, André M. Deelder2, Oleg A. Mayboroda2
Chitra
1
Ratnayake ,
Jerald S.
1
Feitelson
1-Beckman Coulter, Inc., Brea, CA 92822, USA
2-Biomolecular Mass Spectrometry Unit, Leiden University Medical Center, The Netherlands
Increase of Peak Capacity in Bare Fused
Silica Capillaries
Increase of Mass Loading
‰CE is a miniaturized technique
Experimental Conditions for CE-ESI-MS
The union of capillary electrophoresis (CE) with electrospray
ionization – mass spectrometry has great potential as the
fundamental properties of each technique are ideally
complementary to each other. While CE separations are usually
performed at very low flow rates, the ESI process is known to
provide enhanced performance with increased sensitivity and
decreased ion suppression at these low flow rates. To take
advantage of these fundamental properties, we have designed a
robust sheathless interface which through a porous region at the tip
allows for the generation of stable electrospray ionization with flow
rates ranging from below 10 nL/min to >340 nL/min. This flow range
enables the use of CE-MS in either the mass or concentrationsensitive regions of the ESI process. Sheathless coupling further
conserves the resolution gained by capillary electrophoresis, by
eliminating the analyte dispersion that results from stepping up the
low flow generated by electoendosmosis to a substantially higher
pressure driven make-up flow utilized in sheathflow approaches. In
this presentation we assess the potential of this platform for the
analysis of peptide samples of increasing complexity. Particular
attention has been dedicated to parameters such as sensitivity and
peak capacity. To increase the mass loading abilities of the
platform, various online preconcentration methodologies have been
integrated, providing concentration sensitivity down to the low
picomolar level while achieving peak capacities above 300.
Bruker UHR-QTOF MaXis
Distance between porous tip and MS from 3 to 8 mm
ESI voltage between 750 - 1750 V
Wide scanning range
300000
‰Classical Capillary Zone Electrophoresis
9 Sample volume ≤1% Vtot
‰Purpose of integrating preconcentration techniques
9 Increase mass loading without decreasing peak
efficiency
- The BGE is a well-known terminating electrolyte (CH3COOH)
- Sample diluted in ammonium acetate (ammonium as LI)
Experimental Conditions: Vacuum applied at the inlet was varied as follow. (A) 0 psi. (B) 0.5 psi. (C) 1 psi. (D) 1.5
psi. (E) 2 psi. Injection volume = 200 nL (~30% Vtot)
Infusion of an Angiotensin 1 solution (2 μM in 10% acetic acid)
250000
Sheathless Interfacing of CE and ESI-MS
through a Porous Tip
9 Separation capillary – open tube of Vtot <1 μL
Transient Isotachophoresis (t-ITP)
What are the Accessible Flow Rates?
Peak Intensity (Counts)
Abstract
PA 800 plus Capillary Electrophoresis system
30 mm ID bare fused, positively or neutrally coated capillaries (85<L<100 cm)
Inlet OD: 150 μm, outlet OD ~40 μm
10% acetic acid (pH=2.2) or 0.1% formic acid as background electrolytes
Electric field ranging from 300 to 350 V/cm
Generated current between 1 and 5 mA
Sample introduction: hydrodynamic or electrokinetic injection
Use of vacuum in a bare fused silica capillary
(Tryptic digest of BSA at 20 nM)
+
S1 S2 S3 S4
-
LI
200000
Electric Field
150000
Conductivity
‰ Transient formation of a conductivity gradient
¾ All boundaries are stabilized
¾ Large sample zones can be injected
100000
50000
Vacuum at the inlet can be used to tune peak capacity without affecting the
achievable efficiency
Use of Neutral Capillaries
3.0
2.5
Sample: E. coli tryptic digest at 0.5 mg/mL in 50 mM LE
0
0
Stainless steel
cylinder filled
with BGE
BGE
50
100
300
350
t-ITP-CZE Separation
EIEs of 3 peptides obtained by analyzing a BSA tryptic
digest. Injection volume = 200 nL (~30% Vtot , 250 amol
injected). BSA tryptic digest concentration = 1.25 10-9 mol.L-1
0
6 nL injected (1% of the total capillary volume)
Intense. X 10 5
20
30
40
50
60
4
Peak Capacity > 320
3
Conclusion
2
1
0
2.5
5.0
7.5
10.0
70
80
Time [min]
9 Average peak width at half peak height of 11 seconds, 60 min separation window
0
Conductive Housing
(HV Contact)
10
‰ 15 peptides arbitrarily chosen migrating between 26 min and 79 min
6 fmol of BSA tryptic digest injected (1 μM)
Electrospray Tip
1.0
0.0
Bare fused silica capillary, 10% acetic acid BGE
Implementation
+ 34.5 mbar at the inlet (4.2 nL/min following Poiseuille)
0.5
Base peak electropherogram
5
42 nL, 6.2% Vtot, 21 ng loaded,
2.0
1.5
CZE Separation
Inlet for filling the
stainless steel cylinder
with BGE
Static Conductive
Liquid Inlet
250
9 Stable spray between 4 nL/min and 330 nL/min
9 Two different behaviors observed
• “Concentration sensitive:” flow rate >30 nL/min
• “Mass sensitive:” flow rate <30 nL/min
Porous section
Separation
Capillary
200
Flow Rate (nL/min)
ESI voltage
Protective
retractable cover
150
12.5
15.0
17.5
20.0
Depending on the peptide: 2 nM < LOD < 10 nM
(12 - 60 amol injected)
Time [min]
Depending on the peptide: 70 pM < LOD < 210 pM
(14 - 42 amol injected)
The intrinsic properties of the interface allows the operation of the system either in the
concentration-sensitive or mass-sensitive range of the ESI process, resulting in the
achievement of exquisite sensitivities. Preconcentration technique such as t-ITP can easily be
integrated to improve the mass loading of the platform. Sample plugs as long as one third of the
separation capillary (>200 nL) can be used without significantly affecting the achievable
resolution. Depending on whether t-ITP is used or not, the platform provides concentration
limits of detection in the low nanomolar to subnanomolar range, respectively. Finally, it was
demonstrated that the intrinsic properties of the interface even permit the use of non-charged
neutral capillary coatings that exhibit only minute EOF. With this approach we demonstrated
that a very high peak capacity can be reached together with a very high sensitivity, ideal for the
analysis of very complex samples only available in minute amounts