AbstractThis Application Note describes an orthogonal approach to liquid chromatography/mass spectrometry (LC/MS) using an Agilent capillary electrophoresis (CE) system coupled to an Agilent triple quadrupole mass spectrometer for peptide quantifi cation. The newly introduced Agilent Jet Stream (AJS) compatible CE/MS triple tube sprayer and the latest version of MassHunter software (B.05.01), which integrates the control of both CE and MS into a single software platform, have been showcased in this study. Two human serum albumin (HSA) peptides spiked into a tryptic E. coli digest have been used as model compounds to demonstrate the CE/MS/MS multiple reaction monitoring (MRM) approach for quantifi cation. With the CE/MS/MS method, peptides of closely related charge states were separated and attomole detection level was achieved.

CE/MS/MS as an Orthogonal Technique for Sensitive and Easy Quantifi cation of Peptides in Complex Matrixes

Application Note

AuthorsSuresh Babu CV and Srividya KailasamAgilent Technologies, Inc. Bangalore, India

Proteomics

2

sheath-liquid interface (G1607B). The MS was equipped with an Agilent Jet Stream source. The CE/MS system was controlled by Agilent MassHunter Acquisition Software (B.05.01). The sheath liquid was delivered by an Agilent 1200 series Isocratic Pump equipped with a 1:100 fl ow splitter. Agilent MassHunter Peptide Optimizer Software was used to optimize the qualifi er and quantifi er MRM transitions for HSA peptides. The quantifi er was used to generate calibration curves using Agilent MassHunter Quantitative Software. Tables 1 and 2 show the CE/MS parameters and MRM conditions respectively. The approximate amount of sample injected into the separation capillary was calculated using the standard equation for CE.  

ExperimentalMaterialsE. coli total protein lysate was purchased from Bio-Rad. Human serum albumin (HSA) Peptides Standard Mix (G245585001), CE grade water (5062-8578), fused silica capillary (G1600-67311), and sample vials (5183-4623) were obtained from Agilent Technologies, Inc. Bovine serum albumin (BSA) and all other chemicals were purchased from Sigma-Aldrich.

Instrumentation and conditionsAll analyses were performed using the Agilent 7100 CE system coupled to an Agilent 6490 Triple Quadrupole Mass Spectrometer through a triple tube

IntroductionElectrophoretic separation by CE provides an orthogonal tool complimenting liquid chromatographic separation and enhancing coverage and confi dence in the results. As CE is an effi cient and rapid separation technique, it is increasingly being adopted for the analysis of biological samples such as peptides. Growing interest in this technique has led to a number of developments in the instrument hardware and software to enhance user-friendliness. When coupled with tandem mass spectrometry (MS/MS), it becomes an ideal tool for characterization of biomolecules. Increasingly, CE/MS/MS is also being investigated for quantifi cation of these molecules such as peptides1. The recently introduced CE/MS triple tube sprayer is compatible with the AJS source and therefore enhances the sensitivity of analysis of proteins and peptides in complex matrix.

MassHunter acquisition software (B.05.01), is compatible with any of the Agilent TOF, Q-TOF, or Triple Quadrupole CE/MS platforms and integrates control of both CE and MS in a single software. In addition, MassHunter acquisition software for CE/MS/MS incorporates all features of the ChemStation software previously used for CE control. With this new development, it is now easy to switch between an LC/MS or CE/MS setup.

This Application Note demonstrates the performance of CE/MS/MS with the new CE/MS sprayer for the quantifi cation of two human serum albumin (HSA) peptides. In addition, we have shown monitoring 60 MRM transitions for bovine serum albumin (BSA) tryptic peptides within a run time of 35 minutes.

Table 1. CE/MS/MS parameters.

Agilent Capillary Electrophoresis (CE)CE Agilent 7100 Capillary Electrophoresis SystemSample HSA peptide mix Injection 10 s at 50 mbar Capillary Fused silica capillary, total length 60 cm, 50 μm idBuffer 1 M acetic acid, pH 2.3Voltage 30 kV Temperature 20 °CAgilent Triple Quadrupole Mass Spectrometry (MS/MS)MS Agilent 6490 Triple Quadrupole MSIonization mode AJS (positive mode)Acquisition mode MRMSheath liquid 1:1 methanol:water + 0.5 % acetic acid, 8 μL/minDrying gas fl ow 11 L/minDrying gas temperature 200 °CNebulizer 30 psiSheath gas temperature 170 °CSheath gas fl ow 7 L/miniFunnel RF voltage High pressure 150 V

Low pressure 60 VVcap 3,500 VNozzle voltage 300 V

3

Sample preparationFor the calibration curve experiments, stock solutions of HSA peptide standards were made in water at 20 pmol/µL, then serially diluted to the respective concentrations. In the spiking experiments, different concentrations of HSA peptides were spiked into a tryptic digest2 of E. coli. A tryptic digest of BSA was generated as described2 and MRM transitions for the tryptic peptides were generated using Skyline software.

Results and DiscussionThe CE/MS AJS sprayer was developed specifi cally for CE fl ow rates and has a modifi ed sprayer tip. The performance of this AJS compatible CE/MS sprayer has been shown to provide enhanced sensitivity as compared to the standard CE/MS sprayer3.

To demonstrate the CE/MS/MS MRM approach for quantifi cation, two synthetic peptides derived from HSA were chosen as model compounds. Two MRM transitions were monitored for each of the two peptides. Figure 1 shows the overlay of CE/MS/MS MRM quantifi er transitions of the two HSA peptides. Peptide 1 (YLYEIAR) migrates at 3.8 minutes and peptide 2 (LVNEVTEFAK) migrates at 3.9 minutes. The close migration times of the two peptides results from a minimal mobility difference between the two under the acidic CE electrolyte conditions (pH 2.3) employed in the experiment. The use of unique MRM quantifi er transitions for each of the two peptides enables their simultaneous quantifi cation under the present conditions. The qualifi er transitions helped confi rm the identities

Table 2. CE/MS/MS MRM parameters.

Compound Qual/QuantPrecursor ion

MS1 Res

Product ion

MS2 Res Dwell CE

Cell accelerator voltage

YLYEIAR Quantifi er 464.3 Wide 651.0 Unit 50 10 5Qualifi er 464.3 Wide 136.0 Unit 50 10 5

LVNEVTEFAK Quantifi er 575.3 Wide 213.0 Unit 50 15 5Qualifi er 575.3 Wide 937.0 Unit 50 15 5

Figure 1. Overlaid CE/MS/MS MRM electropherograms for HSA peptides: YLYEIAR and LVNEVTEFAK.

×104

0

1

2

3

4

Acquisition time (min)3.2 3.4 3.6 3.8 4.0 4.2 4.4

Coun

ts

YLYEIAR

LVNEVTEFAK

of the two peptides. The MRM transitions selected for peptide 1 (464.3 & 136) and peptide 2 (575.3 & 213) were used in the present study as they provided superior sensitivity under the CE/MS/MS conditions employed. However, these low m/z transitions lack specifi city and might not be the best transitions in a complex mixture. Although only one MRM transition per peptide was monitored in the present proof-of-concept study, typically three to fi ve MRM transitions per peptide are monitored to ensure specifi city.

4

Calibration curves for the two peptides in aqueous buffer were generated over the concentration range of 0.002 pmol/µL–20 pmol/µL. Both peptides showed excellent linearity, with R2 > 0.999, as seen in the log-log plots (Figures 2A, 2B). The data demonstrates a wide linear dynamic range of up to four orders of magnitude. Under the experimental conditions described, for both peptides, the calculated limits of detection (LOD) were found to be 2.3 amol and the limits of quantitation (LOQ) were determined to be 23.5 amol. Figure 3 shows the peaks obtained at the LOD level for the two peptides, as well as overlays of the quantifi er and qualifi er transitions. The LOD of the method was also determined in E. coli digest to ascertain the method performance in a complex matrix. Different concentrations of the HSA peptides were spiked into this matrix and the quantifi er transitions shown in Figure 4 were monitored. The two peptides could be detected at the 2.3 amol level even in this complex background.

Figure 2. Calibration curves of (A) YLYEIAR and (B) LVNEVTEFAK from 0.002 pmol/µL to 20 pmol/µL. Linear fi t with the origin ignored and 1/x weighting.

Peptide 1

Concentration (pmol/µL)0.001 0.005 0.01 0.05 0.1 0.5 1 5 10

0.0001

0.00050.001

0.0050.01

0.050.1

0.51 y = 51,127.127556*x + 83.128892

R2 = 0.9994068658.8 fmol

117.6 fmol235.2 fmol

11.76 fmol

1.176 fmol

23.5 amol

117.6 amol235 amol

Log–log plot

58.8 fmol117.6 fmol

235.2 fmol

11.76 fmol

1.176 fmol

23.5 amol

117.6 amol235 amol

A

B

×106

Peptide 2

Concentration (pmol/µL)0.001 0.005 0.01 0.05 0.1 0.5 1 5 10

0.0001

0.00050.001

0.0050.01

0.050.1

0.51 y = 47,408.413441*x + 58.924210

R2 = 0.99970389Log–log plot

×106

5

+ MRM (575.3 & 213.0) Peptide 2

1 2 3 4 5 6 7

Coun

ts

4.15

4.2

4.25

4.3

4.35

4.4

4.45

4.5

1 2 3 4 5 6 7

Rela

tive

abun

danc

e (%

)

-0.20

0.20.40.60.8

11.21.41.61.8

22.2

575.3 & 213.0, 937.0, 575.3 & 937.0

Ratio = 51.6 (114.8 %)

+ MRM (464.3 & 651.0) Peptide 1

Acquisition time (min)

Acquisition time (min) Acquisition time (min)

Acquisition time (min)1 2 3 4 5 6 7

Coun

ts

4.154.175

4.24.225

4.254.275

4.34.325

4.354.375

4.44.4254.45 *

1 2 3 4 5 6 7Re

lativ

e ab

unda

nce

(%)

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

464.3 & 651.0, 136.0, 464.3 & 136.0Ratio = 82.3 (73.0 %)

Qualifier/QuantifierQuantifier

S/N 4.37

S/N 4.59

A

B

×101

×101 ×102

×102

Figure 3. CE/MS/MS MRM electropherograms of (A) YLYEIAR and (B) LVNEVTEFAK at 2.3 amol.

6

5.0 +MRM (464.3 & 651.0) Peptide 1

4.94.8

Blank

2.3 amol

23 amol

4.74.64.54.44.34.24.1

3.5 3.7 3.9 4.1 4.3 4.5 4.7 4.9

×101

5.0 +MRM (464.3 & 651.0) Peptide 1

4.94.84.74.64.54.44.34.24.1

3.5 3.7 3.9 4.1

Acquisition time (min)

4.3 4.5

S/N 8.39

4.7 4.9

×101

5.0+MRM (464.3 & 651.0) Peptide 1

4.54.03.53.02.52.02.51.00.5

3.5 3.7 3.9 4.1 4.3 4.5

S/N 34.2

4.7 4.9

×107

5.0 +MRM (575.3 & 213.0) Peptide 2

4.94.84.74.64.54.44.34.24.1

3.5 3.7 3.9 4.1 4.3 4.5 4.7 4.9

×101

5.0 +MRM (575.3 & 213.0) Peptide 2

+MRM (575.3 & 213.0) Peptide 2

4.94.84.74.64.54.44.34.24.1

3.5 3.7 3.9 4.1

Acquisition time (min)

4.3 4.5

S/N 5.2

4.7 4.9

×101

5.04.54.03.53.02.52.02.51.00.5

3.5 3.7 3.9 4.1 4.3 4.5

S/N 28.1

4.7 4.9

×107

A B

Figure 4. CE/MS/MS MRM electropherograms of different levels of A) YLYEIAR and B) LVNEVTEFAK in E. coli tryptic digest.

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Further, the capability of the instrument and the software used in the study to handle multiple transitions was shown by analyzing a tryptic digest of BSA. The list of MRM transitions for this experiment was generated using Skyline software4. Figure 5 shows the overlay of 60 MRM transitions for BSA tryptic peptides. The result demonstrates the feasibility of monitoring numerous MRMs in a single CE/MS/MS run. The data quality can be further improved by using dynamic MRM (DMRM) acquisition mode where the peptide transitions are monitored in their respective migration time windows .

ConclusionsThis Application Note demonstrates CE/MS/MS as a sensitive orthogonal approach for peptide quantifi cation over a wide linear dynamic range, even in complex matrixes. The Agilent MassHunter software allows easy control of the CE/MS system and provides the fl exibility to easily switch between LC/MS and CE/MS confi gurations.

0

0.5

1

1.5

2

2.5

3

3.5

Acquisition time (min)

Coun

ts

23 24 25 26 27 28 29 30 31 32 33 34 35

×103

96

Figure 5. Overlay of CE/MS/MS MRM transitions of BSA tryptic peptides. The 60 peptide MRM transitions were imported to MassHunter Acquisition software from a Skyline document and the MRM transition signals were acquired using CE/MS/MS.

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For Research Use Only. Not for use in diagnostic procedures.This information is subject to change without notice.

© Agilent Technologies, Inc., 2014Published in the USA, April 1, 20145991-2729EN

References1. Sun, L., et al., Capillary zone

electrophoresis-multiple reactionmonitoring from 100 pg of RAW 264.7cell lysate digest, Analyst, 2013,138:3181-3188.

2. Glycopeptide Analysis of Antibodiesby Capillary Electrophoresis andQ-TOF Mass Spectrometry, Agilentpublication 5990-7138EN.

3. Compatibility of Agilent Jet Streamthermal gradient focusing technologywith CE/MS, Agilent publication5990-9716EN.

4. MacLean, B., et al., Skyline: an opensource document editor for creatingand analyzing targeted proteomicsexperiments, Bioinformatics, 2010,26(7):966-968.