Ion Trap Mass Spectrometry as Detector for CapillaryElectrochromatography of Peptides: Possibilities and Limitations

Marco Gaspari,1 Marjan Gucek,1, 2 Karin Walhagen,3 Rob J. Vreeken,1ˇElwin R. Verheij,1, 3 Ubbo R. Tjaden,3 Jan van der Greef 1, 3

1 TNO Pharma, Di�ision Analysis and Microbiology, Utrechtseweg 48, NL-3704 AJ Zeist,The Netherlands

2 Faculty of Chemistry and Chemical Technology, Uni�ersity of Ljubljana, Askerce�a 5,ˇ ˇSI-1000 Ljubljana, Slo�enia

3 Leiden�Amsterdam Center for Drug Research, Leiden Uni�ersity, P.O. Box 9502,NL-2300 RA Leiden, The Netherlands

Received 21 February 2001; revised 6 July 2001; accepted 12 July 2001

Abstract: Capillary electrochromatography has been coupled to an ion trap massspectrometer via a sheath flow electrospray interface. Peptide analysis was per-formed on 25 cm columns packed with C -bonded silica particles, and very fast18separations were obtained. Full mass spectrometry and tandem mass spectrometryŽ .MS�MS detection were evaluated in terms of sensitivity and scanning speed. Theseparation of seven selected peptides and their detection by MS�MS in selectedreaction monitoring mode was demonstrated. The performance of this sheath flowinterface also was compared with the previously reported sheathless interface.

Ž .Despite the loss in sensitivity 20�40-fold , the sheath flow interface was shown tobe superior in terms of ruggedness, and allowed the use of higher electric fields toachieve faster analysis times. � 2001 John Wiley & Sons, Inc. J Micro Sep 13: 243�249, 2001

Key words: capillary electrochromatography; peptides; electrospray; ion trap; tandemmass spectrometry

INTRODUCTIONŽ .Capillary electrochromatography CEC is a sep-

aration technique that has experienced a steadygrowth of interest in the past few years. The reasonfor this interest lies in the hybrid nature of thetechnique that brings together the high efficiency ofcapillary electromigration techniques and the ver-satility of high pressure liquid chromatographyŽ .HPLC .

Several reviews have discussed potential andactual applications of the technique, and have oftendrawn parallels between CEC and HPLC or capil-

Ž . � �lary electrophoresis CE 1�3 . In its early stage, thetechnique was mainly used for the separation ofneutral analytes using C or C silica as the station-18 8

Correspondence to: M. Gaspari; e-mail: gaspari@voed-ing.tno.nl.

Present address: Rob J. Vreeken, Micromass Europe,P.O. Box 171, 1380 AD Weesp, The Netherlands.

Contract grant sponsor: Ministry of Science and Tech-nology of the Republic of Slovenia.

Contract grant sponsor: Ferring AB, Malmo, Sweden.

� �ary phase 4,5 . Recent reports are focusing moreand more on the separation of charged compounds� �6�9 and also are stressing the need for new, spe-

� �cially designed CEC stationary phases 10,11 . Theseparation of charged analytes by CEC is even moreinteresting with regard to selectivity, because theseparation mechanism is based on two factors: elec-trophoretic mobility and chromatographic retention.

By far the most common detector used in CECŽ .studies has been an ultraviolet UV detector. In

addition to limited sensitivity and very poor struc-tural information, UV has an additional drawbackwhen used for CEC: it requires that part of thecolumn be free of packing material to allow detec-tion. This discontinuity in the column is believed tobe the major source of air bubble formation, a

� �commonly observed problem in CEC 4,5,12 .Ž .Mass spectrometry MS , although it is definitely

a more expensive detection technique than UV,offers several remarkable advantages with regard tostructural information, sensitivity, and selectivity.Furthermore, MS obviates the need to have an emptysection in the CEC column, because the detection

Ž . Ž .J. Microcolumn Separations, 13 6 243�249 2001� 2001 John Wiley & Sons, Inc.

243

Gaspari et al.244

can take place immediately after the terminating frit� �13,14 .

Different CEC�MS systems have been describ-� �ed by several groups 15�19 . Eluent transport from

the inlet side to the mass spectrometer has beenŽ .achieved in two different ways: i by mere use of

voltage, with the eluent vial kept at ambient pres-Ž .sure and ii by combined use of high pressure and

voltage. This second approach makes the systemmore similar to HPLC, because a laminar flow isinduced. To distinguish between the two systems,the second will be addressed as pressurized CECŽ .pCEC .

Ž .Fast atom bombardment FAB was the first� �ionization technique used to couple pCEC 20 and

� �CEC 15 to MS. With the advent of electrosprayŽ .ionization ESI , mainly two interface designs have

been reported. These two kinds of interfaces alreadyhave been extensively used in CE�MS, as reported

� �by several reviews 21�23 . The first type makes useof a coaxial sheath liquid that provides electricalcontact between the electrospray needle and the

Ž .CEC column outlet sheath flow interface . In thesecond design, the sheathless interface, no make-upliquid is required, and the electrical contact isachieved by applying a metal coating at the tip ofthe CEC outlet. Advantages and drawbacks of bothapproaches have been reported: whereas the sheathflow interface is superior in terms of robustness and

Žallows more freedom in the choice of the eluent or.CE buffer , the sheathless interface provides higher

� �sensitivity, typically 5�20-fold improvement 19,24 .CEC�MS already has been applied to a wide

variety of compounds, and the different applications� �recently were reviewed 12 . Very interesting fea-

tures of CEC�MS have been explored and are stillunder investigation: the possibility of gradient elu-

� � � �tion 16,17 system automation 11 and the use of� �short columns for fast separations 17,25 .

Few CEC�MS reports concerning peptides have� �been published to date 13,26�28 . Tryptic digest

� �analysis was described by Huang et al. 26,27 usingpCEC�MS. The electric field applied to the columnŽ .typically 200�500 V�cm assisted the eluent flowand improved efficiency and selectivity of analysis.

� �In the work of Schmeer et al. 13 , very high electricŽfields were applied to the short CEC column 1000

.V�cm . These authors demonstrated that contribu-tions of pressure-driven and electro-driven flow arenot additive in CEC, and that at such high electricfields, the influence of applied pressure on eluentflow is negligible. In this particular concern, reportsin the literature are still scarce and somehow contra-dictory, and probably more investigation on the in-fluence of applied pressure in CEC�MS systems isneeded. The use of a nanoelectrospray interface to

combine CEC and MS for peptide analysis was de-� �scribed in a recent work 28 , where very good sensi-

� Ž .tivity concentration level of detection LOD below�7 �10 M was obtained in full scan mode and without

any use of sample stacking techniques.In this work, we describe CEC�ESI-MS analysis

on selected peptides. Full scan MS, full scan tandemŽ .mass spectrometry MS�MS , and selected reaction

Ž .monitoring SRM modes of detection are investi-Ž .gated and compared. Because short columns 25 cm

were used to obtain fast separations, the speed ofMS�MS acquisition is discussed also. A sensitivitycomparison is drawn between the sheath flow inter-face described here and the previously reported

� �sheathless interface 28 .

EXPERIMENTALReagents and materials. Acetonitrile was pur-

Žchased from Biosolve B.V. Valkenswaard, The.Netherlands and water was purified through an

Ž .ELGA system Buchs, England , whereas ammo-Žnium acetate was purchased from Sigma Deisenho-

.fen, Germany . Methanol and acetic acid were fromŽ .Merck Darmstadt, Germany . Oxytocin, carbetocin,

desmopressin, and peptide A were kindly suppliedŽ .by Ferring AB Malmo, Sweden . The other peptides¨

Ž .used were from Labkemi AB Stockholm, Sweden .The peptides were dissolved in water to give 1mg�mL stock solutions that were further diluted inthe eluent to give the desired concentration. Thesequences and molecular masses of the peptides arelisted in Table I.

CEC. The separations were performed usingŽ .eluent consisting of 50% v�v acetonitrile in water

with an overall concentration of ammonium acetateof 3.2 mmol�L. The pH of the aqueous ammoniumacetate solution was 6.7. The eluent and the sheathflow liquid were freshly prepared every day and

Žprior to use were thoroughly sonicated at least.15 min . The eluent vial was replenished every

two�three analyses.ŽCommercial CEC columns 100 �m id, 375 �m

.od, 25 cm long , packed with 3 �m Hypersil CECC -bonded silica particles, were kindly supplied by18

Ž .Agilent Technologies Waldbronn, Germany . Eachnew CEC column first was equilibrated with eluentby means of an HPLC pump model 300C from

Ž .Gynkotek Munich, Germany . Then the column wasconnected to the interface, the inlet end of thecapillary was immersed in the eluent, and condition-ing was performed for 10�15 min at 5�10 kV withthe outlet end covered with a droplet of eluent or,alternatively, with the electrospray voltage on. A fewminutes of conditioning also were performed beforeevery injection. After prolonged use of the column,

Capillary Electrochromatography of Peptides 245

Table I. Amino acid sequence of the peptides analyzed and their corresponding molecular mass.aPeptide Amino acid sequence MW

VYV H-Val-Tyr-Val-OH 379.55� �Met -enkephalin H-Tyr-Gly-Gly-Phe-Met-OH 573.75� �Leu -enkephalin H-Tyr-Gly-Gly-Phe-Leu-OH 555.6

Desmopressin Mpa-Tyr-Phe-Gln-Asn-Cys-Pro- Arg-Gly-NH 1069.2D 2

Ž .Carbetocin Bua-Tyr OMe -Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 988.22

Oxytocin H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 1007.22

Peptide A Mpa-Tyr-Phe-� Glu-Asn-Cys-Pro- Arg-Gly-NH 1070.2D 2

a Mpa is mercaptopropionic acid; Bua is butyric acid.

air bubble formation eventually lead to a drop inCEC current and consequently a drop in CEC per-formance; when this happened, the column againwas connected to the HPLC pump and flushed witheluent for 15 min.

To perform CEC, a box kindly provided by theŽ .University of Wales Swansea, UK was used. A

detailed description of the prototype is found else-� �where 19 . No pressurization of the inlet vial was

made, so CEC was run under ambient pressure. Thehigh voltage power supply was from SpellmanŽ .Hauppauge, NY . Injections were performed elec-trokinetically, always with the electrospray voltageoff.

Sheath flow interface MS. All the mass spectro-metric measurements were conducted on an LCQ

Ž .ion trap Thermoquest, San Jose, CA mounted witha nanoelectrospray x-y-z positioner from ProtanaŽ .Odense, Denmark instead of the conventional ESIinterface. A sheath liquid interface to connect theCEC column to the mass spectrometer was builtin-house; it had been used previously to couple

Ž . � �capillary electrophoresis CE to MS 29 . The CEC

column outlet was inserted into a metal needle insuch a way that the outlet of the column extruded0.5�1 mm outside the needle. The position of thecolumn outlet was found to be crucial to achieveoptimum sensitivity. A coaxial sheath liquid flow was

Ždelivered by a Harvard pump Harvard Apparatus,.Holliston, MA at a rate of 1�1.5 �L�min and no

sheath gas was required to achieve a stable spray.The sheath liquid was prepared by mixing MeOH�H O�AcOH in volume proportion of 80�20�0.1.2The x-y-z positioner allowed manual adjustment ofthe distance between the electrospray metal needleand the mass spectrometer sampling capillary toabout 1 cm.

The MS settings for peptide analysis were op-timized with continuous electroinfusion of a 10�mol�L peptide A solution through a nonpackedfused silica capillary. Typical values of the settingswere 175�C sampling capillary temperature, 3 Vsampling capillary voltage, and 3.5�3.8 kV electro-spray voltage. The mass spectrometer was run inpositive ion mode. A schematic view of the instru-mental setup is presented in Figure 1.

CE/CEC box

Inlet vial

Sheath liquid

Mass analyser

MS inlet

ESI stainlesssteel needle

CEC column

HV power

supply

ESI HV

Figure 1. Instrumental setup.

Gaspari et al.246

RESULTS AND DISCUSSIONThe object of this study was to evaluate both the

performance of the sheath flow interface and theability of the ion trap to deal with the high efficiencyof fast CEC separations. The use of short columnsto achieve fast CEC separations is one active area ofCEC research, as mentioned in the Introduction.Nevertheless, commercial instruments, mainly de-signed for capillary electrophoresis, require longcapillaries for connection to the mass spectrometerŽ .about 60�100 cm . For this reason, an importantadvantage of the CE�CEC box used in this workover commercial instrumentation is the possibility to

Ž .accommodate relatively short columns 25 cm . Thisoption allows application of very high electric fieldsto enable short analysis time. Unfortunately, thesetup lacks automation, so no overnight runs couldbe performed and no kind of systematic quantitationstudy was undertaken. Nevertheless, the reliabilityof the system suggests that automation could beachieved efficiently.

Figure 2 depicts examples of full scan MS andfull scan MS�MS electrochromatograms: the threeinjected peptides eluted out of the column in lessthan 5 min. This solution of three peptides was usedas a test mixture to check the performance of thecolumn at regular intervals, using the standard con-ditions described in the legend of Figure 2. Becauseof this, sufficient data on between-day variation ofretention times were collected. As shown in TableII, reproducibility was acceptable, especially consid-ering that injection, voltage ramping, and start ofMS acquisition were performed manually. Anothermajor source of error in the reproducibility of theretention time was the fact that the CEC currentwas always fluctuating within a certain range of

Ž .values typically 5�6 �A , probably due to the factthat the system was run at ambient pressure. At theleast, Table II can be taken as an example to illus-trate the ruggedness of this interface, especially

� �compared with the nanoelectrospray interface 28 .Troubleshooting was reduced to minimum and thesystem always could be started in a reproduciblemanner at the beginning of the day.

It is well known that MS�MS can be used forstructural elucidation of unknowns, but it is usedalso to increase sensitivity of detection for knowncompounds. Under low-energy collision-induced dis-

Ž .sociation CID , peptides fragment mainly along the� �peptide backbone 30 . The two MS�MS spectra

shown in Figure 3 represent the fragmentation of� 5 �Met -enkephalin and oxytocin. The latter is a cyclicpeptide that has a disulfide bond between Cys1 andCys6. This characteristic influences its behavior un-der CID conditions; no fragmentation from the N-

Rel

ativ

e A

bund

ance

2.0 3.0 4.0 5.0 6.0Time (min)

0

100

0

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0

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0

100

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100

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100

0

100

0

100

2.95

4.023.59

2.95

3.59

4.02

Base Peakelectropherogram

Peptide A [M+H]+=1070.6

[Met5]-Enkephalin[M+H]+=574.4

Oxytocin[M+H]+=1007.4

a

2.0 3.0 4.0 5.0 6.0Time (min)

Rel

ativ

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bund

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3.234.90

3.70

3.23

3.70

4.90

Peptide A, MS/MS on m/z=1070.6

[Met5]-Enkephalin, MS/MS on m/z=574.4

Oxytocin, MS/MS on m/z=1007.4

Tic

b

Figure 2. CEC�MS electrochromatogram of a test( )mixture of three peptides 10 �mol�L each in eluent ;

( )electrokinetic injection 10 s at 10 kV , run at 25 kV;( )ESI �oltage 3.8 kV; sheath flow 1.5 �L�min. a Full

( )scan MS; b full scan MS�MS.

Table II. Between-day �ariation of retention timefor three peptides calculated o�er 1 week of analyses( )18 replicates in total ; conditions as in Figure 2.

Average RSD ofretention retention

Ž . Ž .Peptide time min time %

Peptide A 3.21 6.15� �Met -enkephalin 3.72 4.4

Oxytocin 4.63 9.4

Capillary Electrochromatography of Peptides 247

m/z200 250 300 350 400 450 500 550 6000

25

50

75

100R

elat

ive

Abu

ndan

ce397.1

425.0

278.0

380.1 556.2323.1262.0

221.0297.1 480.1 528.2354.0204.9

b5

b3

a4*

a4

b4

a5b2b2

* y2 y3

411.1y4

a

b9

300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

m/z

0

25

50

75

100

Rel

ativ

e A

bund

ance

723.2

990.3

706.2 973.3

933.2916.3

805.2678.1593.0

578.0

644.4412.2

820.3

b6

b7

b8

b9*

b7 *

b8*

b6*

a6 *

b

( ) [ 5] ( )Figure 3. MS�MS spectra of a Met -enkephalin and b oxytocin.

terminal amino acid occurs, so basically only b ionsare present.

Figure 2b shows an electrochromatogram ob-tained using full MS�MS detection. The drawbackof full scan MS�MS detection is the relatively longscan time. The speed of data acquisition is a criticalissue with regard to electromigration techniques,because typical peak widths are on the order of afew seconds when the analysis time is only a fewminutes. If many analytes have to be monitored byMS�MS, the scan cycle can become impracticably

long. A solution is the use of time windows, in otherwords, time-dependent scans. However, time-depen-dent scans applied to such fast separations requireextreme reproducibility of retention time, and this isstill not the case for the system described here.Another MS�MS option, which has been chosen toperform these analyses, is SRM, which is faster thanfull scan MS�MS. If three product ions are moni-tored, the scan time for this MS�MS mode is about0.5 s for each parent ion analyzed. Table III showsthe product ions monitored for each peptide.

Table III. Product ions monitored for each peptide during MS�MS experiments.�� �Peptide M � H Product ions monitored

�Ž . Ž . Ž� � .Peptide A 1070.6 743.2 b , 996.3 b , 1042.4 M � H�CO6 85� � Ž . Ž . Ž .Met -enkephalin 574.4 278.0 b , 397.2 a , 425.0 b3 4 4

�Ž . Ž . Ž .Oxytocin 1007.5 723.2 b , 973.2 b , 990.3 b6 9 9�Ž . Ž . Ž .Carbetocin 988.5 659.2 a , 704.2 b , 914.3 b6 6 8

Ž . Ž . Ž .Tripeptide VYV 380.4 235.0 a , 263.0 b , 281.0 y2 2 2�Ž . Ž . Ž� � .Desmopressin 1069.6 742.2 b , 995.3 b , 1052.4 M � H�NH36 8

5� � Ž . Ž . Ž .Leu -enkephalin 556.5 397.0 a , 425.0 b , 538.1 b4 4 5

Gaspari et al.248

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

Time (min)

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Rel

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2.875.39

3.25

3.49

3.60

3.63

3.93

5.36

*Peptide A

Desmopressin

Oxytocin

[Met5]-Enkephalin

Carbetocin

Leu-Enkephalin

Tripeptide VYV

Figure 4. Selected ion electropherograms of a CEC�(MS analysis of a mixture of se�en peptides 10 �mol�

) (L each in eluent ; electrokinetic injection 10 s at 10)kV ; ESI �oltage 3.8 kV; sheath flow rate 1.5 �L�min;

(� )run at 25 kV. The peak denoted by the asterisk isan interference from desmopressin, because it has justone mass unit difference with peptide A.

Figure 4 shows the analysis of seven peptidesperformed with detection in the SRM mode. It iswell known that CEC separations of charged ana-lytes are due to both electromigration and chro-matographic retention, which include two differentphenomena: hydrophobic interaction and silanophilicinteraction. The latter is defined as a strong electro-static interaction that takes place between positivelycharged analytes and the residual free silanol groupsof the silica particles that start to be negativelycharged above pH 2.5. Whereas the Hypersil CECreversed phase material has a high silanol activityneeded to establish a high electro-osmotic flowŽ .EOF , silanophilic interaction can play an impor-tant role in the separation of peptides, as can beseen in Figure 4. The first peptide that elutes isPeptide A, which is a polar peptide that has atheoretical isoelectric point of 6.3. Peptide A is notretained in these conditions, and it migrates in prox-imity to the electroosmotic flow. Carbetocin, the twoenkephalins, and the tripeptide are retained andseparated mainly by hydrophobic interactions withthe stationary phase. It is interesting to notice thatthe two enkephalins are baseline separated: the moreapolar Leu-enkephalin is retained more. The lasttwo peptides are strongly retained by silanophilicinteractions. Even though the two enkephalins, thetripeptide, and peptide A all possess a positively

charged amino group, silanophilic interaction plays amajor role in only the two peptides that possess anet positive charge at pH 7, i.e., desmopressin and

� �oxytocin, as previously shown by Walhagen et al. 6 .Limit of detection. One of the aims of this work

was to evaluate the sensitivity of the sheath flowinterface for CEC-MS coupling and to compare thiswith the previously used nanoelectrospray interface� �28 . First, the LOD in full scan mode was deter-mined. As shown in Figure 5a, the concentrationLOD is about 2 �mol�L for each of the threeinjected peptides. The corresponding signal to noise

Ž .ratios S�N obtained were 9, 3, and 6 for peptide A,� 5 �Met -enkephalin, and oxytocin, respectively. In ourprevious report on CEC�nESI-MS, a 100 nmol�Lsolution of the same peptides could be analyzed,with even better S�N ratios, and this leads us toconclude that the nanoelectrospray interface is 20�40 times more sensitive than the sheath flow inter-face concerning the analytes of interest. A previous

� �report on CE�MS of peptides 24 indicated a 5�20-fold improvement in sensitivity for most peptidesanalyzed by the sheathless approach compared tothe sheath flow interface.

Figure 5b shows the electrochromatogram of thesame mixture of three peptides, but at a concentra-tion of 500 nmol�L, detected in SRM. The complexfragmentation of this type of analytes caused theoriginal signal to be distributed over a large numberof m�z values, resulting in relevant signal loss ifonly three product ions are monitored. Therefore,only a 4�5-fold improvement in sensitivity was ob-served, whereas typical gains in sensitivity of MS�MSover full scan MS are 1�2 orders of magnitude. Thepeptide that benefits most from MS�MS detection is� 5 �Met -enkephalin, because it has a simpler MS�MS

Žspectrum compared to the other two peptides see.Figure 3 .

In the previous report about nanoelectrospray� �interfacing 28 , we pointed out that maximum sensi-

tivity was obtained at low operating voltages; thebest performances were achieved if the system oper-ated at CEC currents below 2 �A, with electric fieldstrengths not higher than 500 V�cm. Several expla-nations were proposed to explain this behavior, suchas insufficient flow to sustain the spray or air bubbleformation at the electrospray tip. In any case, thisphenomenon was not observed using the sheath flowinterface, so the setup could bear electric fields upto 1000 V�cm and CEC currents in the range 15�20 �A without showing a decrease in performance.The fact that no limitation on electric field strengthis imposed by sensitivity issues is definitely an ad-vantage of the sheath flow interface from the pointof view of speed, because shorter analysis times canbe reached by using higher applied voltages.

Capillary Electrochromatography of Peptides 249

2.0 3.0 4.0 5.0 6.0Time (min)

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100 2.94

3.53

4.15

Peptide A

[Met5]-Enkephalin

Oxytocin

2.0 3.0 4.0 5.0 6.0Time (min)

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100 3.08

3.89

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Peptide A

[Met5]-Enkephalin

Oxytocin

a

b

Figure 5. Analysis at the LOD for a three peptide( )mixture selected ion electropherograms shown ; run at

25 kV; ESI �oltage 3.8 kV; sheath flow rate 1.5 �L�( )min. a Concentration 2 �mol�L, full scan mode;

( )b concentration 500 nM, MS�MS in SRM mode.

ACKNOWLEDGMENTSWe thank Dr. G. P. Rozing at Agilent Technolo-

gies, Waldbronn, Germany, for supplying us withCEC columns.

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