Pulsed Multiple Reaction Monitoring Mode: The Novel Sensitive Approach for Biomolecule Quantitation

Mikhail Belov, Satendra Prasad, David Prior, William Danielson, Karl Weitz, Vladislav Petyuk, Yehia Ibrahim and Richard SmithPacific Northwest National Laboratory, Richland, WA

Introduction

OverviewMethods Results

AcknowledgementsPortions of this work were supported by the National Center for Research Resources, grant RR 18522 and National Cancer Institute, grant R33 CA12619-01. Samples were analyzed using capabilities developed under the support of the NIH National Center for Research Resources (RR18522) and the U.S. Department of Energy Biological and Environmental Research (DOE/BER).

Significant portions of the work were performed in the Environmental Molecular Science Laboratory, a DOE/BER national scientific user facility at Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is operated for the DOE by Battelle under contract DE-AC05-76RLO-1830.

References1. Belov, M.E.; Gorshkov, M. V.; Udseth, H. R.; Anderson, G. A.; Tolmachev,

A. V.; Prior, D. C.; Harkewicz, R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2000, 11, 19-23

2. Wouters, E. R.; Splendore, M.; Mullen, C.; Schwartz, J. C.; Senko, M. W.; Dunyach, J. J. 57th Conference of American Society for Mass Spectrometry; 2009.

3. Ibrahim, Y.; Belov, M. E.; Tolmachev, A. V.; Prior, D. C.; Smith, R. D. Anal. Chem., 2007, 79, 7845-7852.

4. Clowers, B.H.; Ibrahim, Y.M.; Prior, D.C., Danielson, W.F., Belov, M.E.; Smith R.D. Anal. Chem., 2008, 80, 612-623.

Conclusions

CONTACT: Mikhail Belov, Ph.D.Biological Sciences Division, K8-98Pacific Northwest National LaboratoryP.O. Box 999, Richland, WA 99352E-mail: mikhail.belov@pnl.gov

• This work reports on the improved limit of detection of a liquid chromatography (LC)-triple quadrupole instrument operating in the multiple reaction monitoring mode (MRM) by incorporation of an ion funnel trap (IFT) between an ion source and a quadrupole analyzer.

• Incorporation of an ion funnel trap ( IFT) into a triple quadrupole analyzer operating in the multiple reaction monitoring mode (MRM) resulted in 3 to 10 fold improvement in the limit of detection (LOD) as compared to the ion funnel interface and 20 to 50 fold LOD improvement in comparison to that of the commercial instrument.

• Rigorous studies of signal intensities of peptides added to a highly complex biological matrix at concentrations ranging from 0.5 nM to 1000 nM showed the linear response of the LC-IFT-MRM instrument with respect to the concentrations of low abundance peptides.

Multiple Reaction Monitoring (MRM) offers a highly sensitive analytical platform to quantify trace constituents in complex biological matrices by selectively delivering analyte ions from an ESI source to an MS detector. Further sensitivity improvements with LC-MRM approach are achievable by enabling higher efficiency transport of analyte components from an ion source to the MS analyzer, eliminating dead times in analyses of fragment ions and reducing background ion signals. Electrodynamic ion funnel (IF)1or S-lense2 have been shown to drastically improve MS sensitivity. A recently introduced ion funnel trap (IFT) has been demonstrated to further enhance the limit of detection for both the time-of-flight3 and ion mobility time-of-flight mass spectrometers4.The premise for sensitivity improvement with the IFT coupled to a triple quadrupole instrument is due to: i) ion accumulation in the RF-energized trap, which facilitates improved droplet desolvation manifested in the reduced background ion noise at the detector, ii) enhancement in signal amplitude for a given transition because of an order-of-magnitude increase in the ion charge density per unit time compared to the continuous mode of operation, and iii) the unity duty cycle in signal detection, as the use of the trap eliminates dead times between transitions, which are inevitable with continuous ion streams.This work reports on implementation of LC-IFT-MRM analysis of trace constituents from a complex biological matrix using a commercial triple quadrupole instrument (TSQ, Thermo Fisher Scientific).

Chemicals and Materials: Lyophilized Kemptide, Angiotensin I, Syntide 2, Bradykinin, Leucine and Enkephalin, Dynorphin A Porcine 1-13, Neurotensin, and Fibrinopeptide A were purchased from Sigma-Aldrich (St. Louis, MO). These were serially diluted to prepare concentrations ranging from 0.25 nM to 500 nM in 0.25 mg/mL of tryptic digest of Shewanella oneidensis MR-1 proteins.

Figure 1. Experimental setup and graphical interface of instrument control software for an ion funnel trap

Figure 2. Pulsing sequence of experiment with the ion funnel trap. Ions are accumulated in the trap during dead time and dwell time followed by a 0.5 ms release event. Ion release event is synchronized with the start of Q3 scan.

Parent Ions  

Ion Trans. Frag. Trans. Frag. Trans. Frag. CE

Kemptide386.74

(2+) 409.27 b3-NH3 539.34 a5-NH3 567.33 b5-NH3 25

Angiotensin I 432.90 (3+) 534.27 b4 619.36 a5 647.35 b5 21

Syntide 2503.32

(3+) 283.18 b3 429.28 y4 705.94 y142+ 23

Bradykinin 530.79

(2+) 522.27 y92+-NH3 710.36 y6 807.42 y7 30

Dynorphin A Porcine 1-13535.34

(3+) 455.21 y113+ - NH3 529.70 y13

3+ -NH3 712.68 y123+-NH3 28

Leucine Enkephalin556.28

(+) 278.11 b3 397.19 a4 425.18 b4 22

Neurotensin558.31

(3+) 578.85 y92+ 643.73 y10

2+ 725.90 y112+ 26

Fibrinopeptide A768.85

(2+) 445.25 y5 645.33 y7 1077.53 y11 29

Electronics to control IF and IFT

Source interface

TSQ

4 column HPLC

Timing pulse to synchronize trap release and Q3 scan

Voltages to control trap

Transition 1 Transition 2Deadtime

Deadtime

Deadtime

accumulation accumulation accumulation

eject eject ejectEntrance grid

Exit grid

Transition 3

Q3 Trigger

Exit grid Entrance grid

ION FUNNEL TRAP Q1 Q3Collision cellQ2

Detector

ION FUNNEL

10 torr 1 torr 1.5x10-3 torrmulti inlet

-1

-0.6

-0.2

0.2

0.6

1

0 5 10 15 20 25RETENTION TIME, min

INTE

NSI

TY

ION FUNNEL TRAP2 nM Anigiotensin I in 0.25 mg/mL Shewanella digest

ION FUNNEL 2 nM Anigiotensin I in 0.25 mg/mL Shewanella digest

0 5 10 15 20 25 30 350.000E+00

1.000E+08

2.000E+08

3.000E+08

4.000E+08

5.000E+08

6.000E+08

R² = 0.994070172854722Kemptide

fmoles loaded onto LC column

Peak

Are

a

0 5 10 15 20 25 30 350.00E+00

1.00E+08

2.00E+08

3.00E+08

4.00E+08

5.00E+08

6.00E+08

R² = 0.997333156050591

Angiotensin I

fmoles loaded onto LC column

Peak

Are

a

0 5 10 15 20 25 30 350.00E+00

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

R² = 0.996003925872965Leucine Enkephalin

fmoles loaded onto LC column

Peak

Are

a

Peak

Are

a

0 5 10 15 20 25 30 350.00E+00

1.00E+08

2.00E+08

3.00E+08

4.00E+08

5.00E+08

6.00E+08

7.00E+08

R² = 0.994467325150108

fmoles loaded onto LC column

Fibrinopeptide A

5 10 15 20 25 30 350E+00

1E+08

2E+08

3E+08

4E+08

5E+08

6E+08

R² = 0.998842838158562

Neurotensin

fmoles loaded onto LC column

Peak

Are

a

0 5 10 15 20 25 30 350.00E+00

4.00E+07

8.00E+07

1.20E+08

1.60E+08

2.00E+08

R² = 0.99828682610235

fmoles loaded onto LC column

Bradykinin

Peak

Are

a

Figure 3. LC-MS/MS experiment with a 0.25 mg/mL Shewanalla oneidensis digest spiked with nine peptides (see table 1). Inset shows three transitions of neurotensin acquired in continuous (blue trace) and trapping (red trace) modes

SN

R

SN

R

kemptideLeucine Enkephalin

moles moles

Figure 4. Signal-to-Noise (SNR) of kemptide and leucine enkephalin as a function of the peptide amount added to a 0.25 mg/mL Shewanella oneidensis digest in LC-MRM experiments. Bottom panels show selected ion chromatograms representing three transitions of the two peptides and the matrix signal in the corresponding m/z ranges.

Table 1. List of peptides and the corresponding fragments monitored in LC-MRM experiments with a 0.25 mg/mL Shewanella oneidensis digest

0

20000

40000

60000

80000

534.265 534.266 534.267 534.268

m/z

INTE

NSI

TY

Angiotensin I, b4+, m/z 534.27ION FUNNEL TRAP

ION FUNNEL

0

20000

40000

60000

80000

619.356 619.357 619.358 619.359

m/z

INTE

NSI

TY

Angiotensin I, a5+, m/z 619.36

ION FUNNEL TRAP

ION FUNNEL

0

20000

40000

647.351 647.352 647.353 647.354

m/z

INTE

NSI

TY

Angiotensin I, b5+, m/z 647.36

ION FUNNEL TRAP

ION FUNNEL

Figure 5. Comparison of the selected ion chromatograms corresponding to the three transitions of Angiotensin I added with other 8 peptides to a 0.25 mg/mL digest of Shewanella oneidensis in LC-MRM experiments. Trapping time in the IFT was 40 ms.

Figure 6. Peak area as a function of the peptide amount for several peptides added to a 0.25 mg/mL Shewanella oneidensis digest in LC-IFT-MRM experiments.