Anal. Chem. 1994,66, 2835-2840
Capillary Electrophoresis/Electrospray Mass Spectrometry Using a High-Performance Magnetic Sector Mass Spectrometer John R. Perkins'lt and Kenneth B. Tomer* Kratos Analytical, Barton Dock Road, Urmston, Manchester, M3 1 ZLD, U.K., and National Institute of Environmental Health Sciences, P.0. Box 12233, Research Triangle Park, North Carolina 27709
Capillary electrophoresis/electrospraymass spectrometry using a high-performance magnetic sector mass spectrometer has been performedwith both bare fused silica and 3-(aminopropy1)trimethoxysilane-derivatizedsilica columns for simple peptide mixtures and for a snake venom. The latter is essentially a naturally occurring complex mixture of small proteins. Adaptation of the existing electrospraysystem to accommodate capillary electrophoresismethodologies was extremely simple. Full-scan CE/MS on a sector mass spectrometer combined sensitivity with high resolution for discrimination between species of similar mass to charge ratios. Electrospray mass spectrometry (ESI-MS) has generated a great deal of interest due to the suitability of the technique for the analysis of high molecular mass and thermally labile compounds. Frequently, samples presented for mass spectrometric analysis are complex and require a separation step prior to mass analysis. We have been interested in the development of low flow rate (nanoliters per minute) separations in conjunction with mass spectrometry. Chief among these has been capillary electrophoresis, which is characterized by high separation efficiency and high analyte flux. Capillary electrophoresis was initially reported by Jorgenson and Lukacs in 198 1.I Capillary columns (10-1 00 pm i.d.) are filled with buffer solution and a voltage is then applied across the column. The separation of the analytes results from differential migration in the electric field, which depends on the net charge on the analytes at a particular solution pH, with contributions from molecular size and spatial distribution2 CE flow has a flat pistonlike profile in contrast to the parabolic flow profile associated with liquid chromatographic separations. Consequently, the separation efficiencies achieved are extremely high.* Migration times are a function of both the electromigration of the analyte and the bulk electroosmotic flow and can be adjusted by altering the pH or buffer composition or by addition of an organic m ~ d i f i e r . ~ Electroosmotic flow is greater than the analyte electromigration rate and is, thus, usually strong enough to sweep all neutral and charged analytes to ground. A potential problem encountered with basic peptides and proteins is that they retain a net positive charge even at high
* Present address:
16 Sandhurst Rd., Mile End, Stockport, Cheshire, SK2 7NY,
U.K. + Kratos Analytical. t
National Institute of Environmental Health Sciences.
(1) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981.53, 1298-1302. (2) Jorgenson, J. W. Am. Chem. SOC.Symp. Ser. 1987, No. 335, 182. (3) McCormick, R. M. Anal. Chem. 1988, 60, 2322-2328.
0003-2700/94/0366-2835$04.50/0 0 1994 American Chemical Society
pH's which leads to adsorption to the negatively charged column walls and, consequently, to peak broadening3 One approach to counter this problem is the derivatization of the column wall, and the applications of several derivatizing reagents have been r e p ~ r t e d . ~We - ~ have reported the use of columns derivatized with 3-(aminopropyl)trimetho~ysilane.~~~~ Due to the addition of aminopropyldimethoxy groups to the column surface, the charge on the column wall becomes positive. This causes the direction of the electroosmotic flow to change, since it is driven by an excess of negative ions in solution, so that it flows from a high negative potential to ground.12 Use of buffers below pH 10.6 means that the wall will have a positive charge and will repel positively charged analytes.11J4 Mass spectrometry provides a desirable detection system for capillary electrophoresis because it is almost universal and sensitive and does not require previous derivatization. Additionally, the information provided by mass spectrometry means that reproducible migration times obtained through rigid control of the C E system are not necessary for compound identifi~ati0n.l~ To date C E has been interfaced directly with mass spectrometry via fast atom bombardment and electrospray ionization and indirectly via 252Cf plasma desorption and matrix-assisted laser desorption ionization mass spectrometry.14 CE was initially coupled with electrospray mass spectrometry in 1987,15where the on-line separation of quaternary ammonium salts, nucleosides, and peptides was demonstrated. In these experiments, the total column effluent was introduced to the mass spectrometer with no additional make-up f l o ~ . ~ ~ J ~ Electrospray requires a minimum flow greater than the standard operating flow rates of CE17 so subsequent (4) Jorgenson, J. W.; Lukacs, K.D. Science 1983, 222, 266-272. (5) Hjerten, S.J. J . Chromatogr. 1985, 347, 191-198. (6) Cobb, K. A.; Dulnik, V.; Novotny, M. Anal. Chem. 1990, 62, 2478-2483. (7) Wiktorowicz, J. E.; Colburn, J. C. Electrophoresis 1990, 11, 769-773. (8) Thibault, P.; Paris, C.; Pleasance, S. Rapid Commun. Mass Spectrom. 1991, 5 , 484-490. (9) Towns, J. K.; Regnier, F. E. J. Chromatogr. 1990, 516, 69-78. (IO) Moseley, M. A.; Deterding, L. J.; Tomer,K.B.; Jorgenson, J. W. Anal. Chem. 1991, 63, 109-114. (11) Moseley, M. A.; Jorgenson, J. W.; Shabanowitz, J.; Hunt, D. F.; Tomer, K. B. J . Am. SOC.Mass Spectrom. 1992, 3, 289-301. (12) Parker, C. E.; Perkins, J. R.; Tomer,K. B.; Shida, Y.; O'Hara, K.; Kono, M. J . Am. SOC.Mass Spectrom. 1992, 3, 563-574. (1 3) Perkins, J. R.; Parker, C. E.; Tomer, K.B. Electrophoresis 1993,14,458-468. (14) Tomer, K. B.; Deterding, L. J.; Parker, C. E. submitted for publication in Adu. Chromatogr. ( N .Y.). (15) Olivares, J.A.;Nguyen,N.T.;Yonker,C.R.;Smith,R. D.Ana/. Chem. 1987, 59, 123g1232. (16) Smith, R. D.; Olivares, J. A.; Nguyen, N. T.; Udseth, H. R. Anal. Chem. 1988, 60, 43-41,
Analytical Chemistry, Vol. 66,No. 18, September 15, 1994 2835
interfaces have employed make-up flow provided by a coaxial applied to commence the electrophoretic separation. Initial flow delivery system11-*4v17-28 or a liquid junction ~ y s t e m ~ * - ~ ~separations were performed on a 1.1 m X 7 5 pm i.d. X 375 for the stable operation of the electrospray and closely related pm 0.d. fused silica column (Polymicro Technologies, Phoenix, gas nebulizer assisted electrospray (ion spray) source. CE/ AZ). Due to the pronounced tailing noted for basic peptides ESI-MS has proven ideal for the analysis of peptides, proteins, on bare fused silica, further experiments employed a 1.1 m tryptic digests, and other compounds of biological imporlength of fused silica which had been derivatized with tance.I4 We have been interested in interfacing CE with a 3-(aminopropy1)trimethoxysilane using a method described magnetic sector mass spectrometer to determine whether the p r e v i ~ u s l y . l @The ~ ~ buffers employed throughout the CE improved resolution could yield additional information and to experiments were 0.01 M ammonium acetate (pH 8) with the test whether we could maintain good separation without underivatized column and 0.01 M acetic acid (pH 3.5) with degradation of data when employing magnet scans. We report the APS-derivatized fused silica. For the experiments studying our results here. snake venoms, the column was flushed with buffer solution between runs. Extensive use of the column could, however, EXPER I MENTAL SECT1ON lead to extended migration times and peak broadening symptomatic of column deactivation. The column could then Capillary Electrophoresis. The CE system was constructed be reactivated by flushing with 0.01 M HC1.35 Separate in-house and has been described e l s e ~ h e r e . It ~ ~utilizes ?~~ a columns were used for FIA and CE of the snake venoms to Glassman HV power supply with reversible polarity which was operated at +38 kV during experiments with the minimize column deactivation prior to CE experiments. underivatized fused silica column or -22 kV with the APSMass Spectrometry. Electrospray mass spectra were derivatized column. The high-voltage end of the CE capillary acquired on a Kratos Concept 1SQ (Kratos Analytical, was located in a plexiglass box while the C E ground was the Urmston, UK) of EBqQ geometry fitted with an electrospray electrospray needle itself, giving an effective voltage drop of ionization source.36 The electrospray needle was operated at -30 kV. a potential of 8 kV and the accelerating potential was 4 kV. The experiments reported here were initially concerned Flow injection analysis of the standard compounds was with assessing the viability of CE/ESI-MS using a highperformed by placing the sample vial in the well of a stainless performance mass spectrometer rather than an attempt to steel pressure vessel, described p r e v i ~ u s l y and , ~ ~ forcing the provide maximum separation efficiency or maximum sensitivanalyte through a silica column (75 pm id., 150 pm 0.d.) by ity. Consequently, initial experiments employed timed injecpressurizing with helium. A make-up solution of 50:50 tions from a stainless steel pressure vessel, which tended to methanol/3% acetic acid was provided coaxially from a lead to overloading and peak degradation for most components. Harvard syringe pump, Model 909 (Harvard Apparatus, South For experiments requiring smaller injection volumes, timed Natick, MA). Flow injection analysis (FIA) spectra were gravimetric injection was employed due to the bias encountered acquired in the profile mode from m/z = 400 to m/z = 2200 using electromigration injection techniques. During injection, at a scan rate of 5 s/decade. CE/ESI-MS of a mixture of the capillary was placed in a sample vial held at a fixed distance bioactive peptides was performed over a mass range of 400above the CE buffer for a timed interval. The end of the 2200 at 1 s/decade while CE/ESI-MS of luteinizing hormone capillary was then placed into the CE buffer and the voltage releasing hormone and related peptides was performed over a mass range of 400-1 500 at 1 s/decade. CE/ESI-MS analysis (17) Smith, R. D.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1988, 60, 1948of the snake venoms was performed over a mass range of 1952. (18) Perkins, J. R.; Parker,C. E.; Tomer, K. B. J.Am. SOC.MassSpectrom. 1992, 400-2200 at 2 s/decade. Mass calibration during all experi3, 139-149. ments was provided by a mixture of gramicidin S and bovine (19) Deterding, L. J.; Parker, C. E.; Perkins, J. R.; Moseley, M. A,; Jorgenson, J. W.; Tomer, K. B. J. Chromatogr. 1991. 554, 329-338. insulin. The mass resolution was 1000 (10%valley definition). (20) Moseley, M. A.; Jorgenson, J. W.; Tomer, K. B.; Hunt, D. F.; Alexander, J. Data acquisition and system control was performed using a E.; McCormack, A. L.; Martino, P. A.; Michel, H.; Shabanowitz, J.; Sherman, N. In Techniques in Protein Chemistry; Villafranca, J . J., Ed.; Academic Sun SPARC station. The theoretical molecular masses of Press: New York, 1991; Vol 11, pp 441-454. peptides and proteins for known sequences were calculated (21) Huang, E. C.; Henion, J. D. Anal. Chem. 1991, 63, 732-739. (22) Loo, J. A.; Jones, H. K.; Udseth, H. R.; Smith, R. D. J. Microcolumn Sep. using MacProMass (Beckman Research Institute City of 1989, 1, 223-229. Hope, Duarte, CA). (23) Loo, J. A.; Udseth, H. R.; Smith, R. D. Anal. Eiochem. 1989,179,404412. (24) Smith, R. D.; Loo, J. A,; Barinaga, C. J.; Edmonds, C. G . ; Udseth, H. R. J. For CE/ESI-MS experiments, the standard Kratos elecChromatogr. 1989, 480, 21 1-232. ( 2 5 ) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth. H. R. J. trospray probe was modified to accommodate a zero dead Chromatogr. 1990, 516, 157-165. volume tee which permitted the coaxial introduction of a make(26) Pleasance, S.; Ayre, W.; Laycock, M. V.; Thibault, P. Rapid Commun. Mass Spectrom. 1992, 6, 14-24. up flow of 50:50 methanol/3% acetic acid (Figure 1). This (27) Thibault, P.; Pleasance, S.; Laycock, M. V. J. Chromatogr. 1991, 542, 483501. provided sufficient liquid to maintain a stable spray without (28) Pleasance, S.; Thibault, P.; Kelly, J. J. Chromatogr. 1992, 591, 325-339. compromising the analytical separation. The electrospray (29) Lee, E. D.; Mueck, W.; Henion, J. D.; Covey,T. R. J. Chromatogr. 1988,458, 31 3-321. needle employed during C E acquisitions was a piece of
(30) Mueck, W. M.; Henion, J . D. J. Chromatogr. 1989, 495, 41-59. (31) Lee, E. D.; Mueck, W.; Henion, J . D.; Covey, T.R. Biomed. Enuiron. Mass Spectrom. 1989, 18, 844-850. (32) Lee, E. D.; Mueck, W.; Henion, J. D.; Covey, T. R. Eiomed. Enuiron. Mass Spectrom. 1989, 18, 253-257. (33) Moseley, M. A.; Deterding, L. J.; Tomer, K. B.; Jorgenson, J. W. Rapid Commun. Mass Spectrom. 1989 3 87-93. (34) Tomer, K. B.; Moseley, M. A. In Continuous Flow Fast Atom Bombardmenf; Caprioli, R.M., Ed.; Wiley & Sons: New York, 1990; pp 121-136.
2836
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(35) Perkins, J. R.; Parker, C. E.; McGown, S. R.; Tomer, K. B. 40th ASMS Conference on Mass Spectrometry and Allied Topics, Washington, DC, June 1-5, 1992; pp 961-962. (36) Chapman, J. R.; Gallagher, R. T.; Barton, E. C.; Curtis, J . M.; Derrick, P. J. Org. Mass Spectrom. 1992, 27, 195-203. (37) Deterding, L. J.; Moseley. M. A.; Tomer, K. B.; Jorgenson, J. W. Anal. Chem. 1989, 61, 2504-2511.
Shah Flow
I h.lyf1C.l
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I
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Nedlea18.5 k v
-
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I
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Flgure 1. Modified probe used during CE/ESI-MS experiments.
. i.
Met-Enkephalin"
I 1
mlz = 573.5
m
Proctolln
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mh=650 I
L
1
I
.~.
Mel.Enkeph.lln-Arg-Phb
mlz = 178
0-Caromorphln
mh = 791
stainless steel tubing 0.028 in. 0.d. X 0.017 in. i.d. (22 gauge). This larger needle permits the use of larger outer diameter (375 . .um) columns which have proved much more resistant to *electro-&illingnloand column breakage. column position within the needle proved crucial for spray stability. Chemicals. The standard samples of the luteinizing hormone releasing hormone and related peptides, a-melanocyte stimulating hormone (a-MSH), proctolin, Met-enkephalinamide, Met-enkephalin-Arg-Phe, 0-casomorphin, neurotensin, 3-(aminopropyl)trimethoxysilane, and the snake venoms were obtained from Sigma Chemical Co. (St. Louis, MO). Artificial peptide mixtures and portions of snake venom (1 mg) were dissolved in water which was obtained from a Milli Ro/Milli-Q system (Millipore Corp., Bedford, MA). The mobile phase employed HPLC grade acetic acid which was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ) while HPLC grade methanol was obtained from Fisher Scientific (Fairlawn, NJ).
RESULTS AND DISCUSSION Our premier concern was the extent to which the electrospray system was affected by a large positive voltage at the injection end of the CE column. During CE/ESI-MS with underivatized fused silica columns, using a quadrupole mass spectrometer, it was noted that when the high voltage of the power supply was applied the voltage on the needle tip increased substantially as a result of conductivity through the column. This could lead to loss of tuning in the electrospray source and was countered by a decrease in the voltage on the electrospray control box. This was not observed with the magnetic sector mass spectrometer, presumably because the needle was already at a high voltage (8 vs 3 kV) and therefore much less likely to be affected. The extracted electropherograms from the full-scan C E separation of a mixture of six bioactive peptides using an ammonium acetate buffer (0.01 M) at pH 8 are illustrated in Figure 2. This corresponds to an injection of 1 pmol of neurotensin to 3 pmol of Met-enkephalinamide on column, and the resultant overloaded peaks are obtained. These extracted electropherograms correspond to the (M + 2H)2+ charge states of neurotensin and a-MSH and the (M H)+ ions of Met-enkephalinamide, proctolin, Met-enkephalin- ArgPhe, and 6-casomorphin. Further optimization of the analysis of this artificial mixture was not pursued. In our laboratories, we have placed significantly greater emphasis on the use of derivatized fused silica capillaries during CE/MS as a result of their superior applicability for separations involving basic peptides and p r ~ t e i n s . ~ - The l~ extracted electropherograms from the full-scan CE/MS separation of five luteinizing hormone releasing hormones (LH-RHs) are illustrated in Figure 3. This corresponds to
+
400
450
500
550
1O:lS
11:35
12:52
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600 15:27
650 16:44
700 18:02
800
750 19:lY
20:36
Figure 2. Extracted electropherograms from CE/ESI-MS of six bioactive peptides using a 1.1-m length of 75-p-i.d., 375-p-0.d. bare fused silica column. The buffer employed was 0.01 M ammonium acetate at PH 8. The signals correspond to on-column injections of 1 pmol of a-MSH (M, = 1685.1) and neurotensin (4 = 1673.2), 1.8 Pmol of Met-enkephalin-R-F (M, = 877.2), 2 pmol of 8-casomorphin (M, = 790.0), 2.5 pmol of proctolin (M, = 848.8), and 3 pmol of Met-enkeDhalinamMe (M = 572.8).
I
1 LH.RH mh-101.8 77%
[LI-Ales]-LM.RH mh = 51.6
m h = 626.6
SCall
Figure 3. Extracted electropherograms from CEIESI-MS of five luteinizing hormone reieasing hormones using a 1.1-m length of 75p-i.d., 375-p-0.d. fused silica column, previously derivatized wRh 3-(aminopropyl)trimethoxysllane.The buffer employedwas 0.01 M acetic acid at pH 3.5. This corresponds to an on-column injection of 1 pmoi of each of the LH-RHs.
an on-column injection of 700 fmol of each component. The migration time of these decapeptides was found to increase with the number of basic residues within their sequences, with [ D - P ~ ~ ~ , D - A ~ ~ ~ ](one L H -basic R H residue) first with a migration time of 11 min 45 s through to [ D - L ~ S ~ I L H - R H at 16 min 51 s. LH-RHand [GlyOHIO]LH-RHwere readily separated because the presence of the free acid at the C-terminus of [GlyOHIO]LH-RHreduced the migration time relative to LH-RH where the amide is present at the C-terminus. It was not possible to separate LH-RH from [D-Ala6]LH-RH because the change in sequences only resulted from the replacement of one nonpolar amino acid (alanine for glycine) with another. Relative to the quadrupole instrument and data system in our laboratory, a perceived advantage of CE/MS on a magnetic sector mass spectrometer was sensitivity. On the quadrupole instrument in the laboratory at NIEHS, the acquisition of full-scan CE/MS data was difficult and in many cases impossible, such that single ion monitoring was employed during most experiments. The approach during the experiments reported here was to employ full-scan CE/MS throughout so that spectra could be used to conclusively identify analytes. Standard spectra of all of the LH-RHs with the
-
AnalyticalChemistry, Vol. 66,No. 18, September 15, 1994
2837
1111
A 1°,i 90
K
Flgure 4. On-line ESI-MS spectra of (A) [Gly-OH'O]LH-RH showing the (M 2H)2+ ion at m/z = 592.2, (B) comigrating [DAlae]LH-RH showing the (M 4- 2H)*+ ion at m/z = 599 and LH-RH showing the (M 2H)2+ion at m/z = 591.8 together with a slight (M H)+ ion at m/z = 1183, and (C) [DLyss]LH-RH showing the (M 2H)2+ion at m/z = 627.
+
+
+
+
exception of [D-Phe2,D - A ~ ~ ~ I L H -(one R Hbasic residue) are dominated by the (M 2H)2+ions, though some slight (M H)+ ions are noted (Figure 4). It was observed during flow injection analysis that the response obtained for [D-Phe2,DAla6]LH-RH was a fraction of that seen for the other luteinizing hormone releasing hormones. Consequently, during CE/MS at the 700-fmol injection level, the spectra of the four other luteinizing hormone releasing hormones were readily identifiable but the spectrum of [D-Phe2.D-Ala6]LHRH was poor (Figure 4). We have recently been interested in the utility of CE/ ESI-MS for the characterization of snake venoms which are essentially naturally occurring complex polypeptide/protein mixtures.I3 Experiments using a quadrupole mass spectrometer were limited by the instrumental mass range of 1200 Da. This prevents the detection of toxins of molecular masses greater than -8000 Da. However, using CE-ESI-SIM-MS we have been able to demonstrate the presence of over 100 toxins in the venom of the black mamba.13 We have acquired the ESI spectra by flow injection analysis on the sector mass spectrometer of eight venoms.38 The ESI spectrum obtained from FIA of the venom from Naja melanoleuca (black and white cobra) is shown in Figure 5. Some of the toxins from the venom of N . melanoleuca have been isolated using gel filtration and ion chromatography with subsequent protein sequencing.3945 The electrospray spectrum acquired to m/z = 2200 allows the detection of toxins in the molecular mass range 6000-9000 Da together with toxins around 13 000 Da. Flow injection analysis suffers due to the suppression noted for the higher molecular mass species38while lower molecular mass peptides show multiplecharge states at higher mlzvalues than are noted in on-line CE/MS spectra (e.g., the peptide of molecular mass 6769.4 Da shows the (M + 6H)6+ ion at m/z = 1130, (M + 5H)5+ion at m/z = 1354, and (M + 4H)4+ at m/z = 1693 under FIA, while it shows the (M 6H)6+ at m/z = 1130 and (M + 5H)5+ at m/z = 1354 using CE/MS). Additionally, a separation step is required before we can
+
+
+
(38) Perkins, J. R.; Tomer, K. B.; Smith, B.; Gallagher, R. T.; Jones, D. S . ; Davis S.C.; Hoffman, A. D. J . Am. SOC.Mass Specirom. 1993, 4, 670-684. (39) Botes, D. P. J . Bioi. Chem. 1972, 247, 2866-2871. (40) Carlsson, F. H. H. Biochem. Biophys. Res. Commun. 1974, 59, 269-276. (41) Carlsson, F. H. H.; Joubert, F. J. Biochim. Biophys. Acfa 1974,336,453469. (42) Joubert, F. J. Biochim. Biophys. Acia 1975, 379, 329-344. (43) Carlsson, F. H. H . Biochim. Biophys. Acra 1975, 400, 31C-321. (44) Joubert, F. J. Biochim. Biophys. Acia 1975, 379, 345-359. (45) van Eijk, J . H.; Verheij, H. M.; De Haas. G. H . Eur. J . Biochem. 1984,139, 51-57.
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Analflical Chemistry, Vol. 66,No. 18, September 15, 1994
Figure 5. ESI-MS spectrum of the venom from N. melanoleuca (black and white cobra)obtainedby flow injectionanalysis. Using this spectrum some of the identified peptides include the following: M, = 6672 [(M 7H)7+= 954, (M 6H)6+ = 1113, (M 5H)5+ = 1335, (M 4H)4+ = 16691; M, = 6769 [(M 6H)O+ = 1130, (M 5H)5+ = 1354, (M 4Hr+ = 16931; M, = 6794 [(M 7H)7+= 972, (M 6H)s+ = 1133, (M 5H)5+ = 1359, (M 4H)'+ = 17001; M, = 13348 [(M 9H)9+ = (M 8H)O+ = 1669, (M 7H)'+ = 19071; M, = 6695 [(M 6H)e+ = 1117, (M 5H)S+ = 1340, (M 4H)'+ = 16741; M, = 6841 [(M 4- 7H)7+ = 978, (M 6H)O+ = 1141.5, (M = 13691.
+ +
+
+
+
+
+
+
+
+
+
+
+
+
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. 60
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Flgure 6. ReconstructedTIC from CE/ESI-MS of the venom from N. melanoleuca (60-ng on-column injection of the total venom).
determine whether any one m/z value relates to more than one peptide or whether multiple peptides are noted with identical molecular mass. This has been known to occur during CE-ESI-SIM-MS experiments.I3J5 Full-scan CE/ESI-MS experiments followed two approaches. The first method utilized a pressure injection so that a large volume of venom was injected. An on-column injection corresponding to 60 ng of venom led to overloading and degradation of the CE separation but provided the most useful spectra for peptide and protein identification. The reconstructed total ion current trace obtained from this separation is shown in Figure 6, and extracted electropherograms of some minor components not seen at lower injection volumes are illustrated in Figure 7. Typical representative full-scan summed spectra of various components are shown in Figure 8. The full value of CE/ESI-MS vs flow injection analysis is illustrated by the two major peptides in the venom, which show molecular mass of 6674 (toxin Viil) and 13349 Da, respectively, such that multiple charged ions arising from each will coincide because the molecular mass of the smaller toxin is approximately half that of the larger toxin. Under flow injection analysis conditions, the mass spectrum seems to be dominated by the peptide of molecular mass 6674 Da with ions at m/z = 954 (M + 7H)'+, m/z = 1113 ( M + 6H)6+, m/z = 1335 (M + 5H)S+,and m/z = 1669 ( M 4H)4+, and
+
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Figure 9. Extracted spectra of the two major toxins from the venom of N. melanoleuca. Toxin Viil is approximatelyhalf the molecular mass of the molecular mass obtained for unresolved phospholipases DE-I I 1 and DE-IIIA so discrimination between the two is only possible after insertion of a separation step.
Figure 7. Extracted electropherograms from some of the minor components of the venom of N. melanoleuca: peak 1, m/z = 1667.5; peak 2, m/z = 1506; peak 3, m/z = 1683; peak 4, m/z = 1268, M, = 6769.4 Da; peak 6, cdk = 6336.0 Da; peak 5, m/Z = 1130, Mr m/z = 1061.5; peak 7, m/z = 1141.5, toxin ViiP.
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Figure 10. Expansion of the (M 8H)8+ and (M -t 7H)'+ regions of the mass spectrum from the peak with average mass of 13 348 Da to reveal the presence of phospholipase DE-I11 and phospholipase DE-IIIA
mh
Figure 8. Typical on-line summed spectra of some toxins obtained from CE/ESI- MS of the venom from N. melanoleuca: (A) toxin of molecular mass 13 308.3 Da; (B) toxin of molecular mass 13 366.6 Da, (C) toxln of molecular mass 7118.8 Da; (D) toxin Vii3.
only ions of low relative abundance at m/z = 1484 and 1906 provide indication of the higher molecular mass peptide. The insertion of a separation step is the only means of discriminating between these toxins. The extracted summed spectra from CE/MS indicate that toxin Viil shows charge states at m/z = 954, 11 13, and 1335 Da, while the higher molecular mass peptide shows charge states at m/z = 1484, 1670, and 1906 Da (Figure 9). The relative responses of the major protonated ions from both spectra are 4300 mV for m/z = 1670 and 3300 mV for m/z = 1113. Further expansion of the spectrum of the peptide of average mass 13 348 Da indicates the peak results from at least two similar peptides (Figure 10). These are phospholipase DE-I11 (Mr= 13 345) and phospholipase DE IIIA ( M r = 13 350.9), which differ in only 2 of 120 amino acids.42 The two peptides have identical PI'S, which indicates that they would show similar electrophoretic mobilities and thus could not be separated by CE. Experimental molecular massesof 13345.7 Da (DE-111) and 13 348.9 (DE-IIIA) were determined for the two phospholipase^.^^ For the most useful CE/ESI-MS separation of the major toxins, an on-column injection corresponding to 16 ng of total venom was employed. The extracted ion electropherograms of the 10 major toxins of N . melanoleuca are illustrated in Figure 1 1. It can be seen that the peaks due to phospholipases
t
15
m/z = 1493 1e
00%
m h = 972 1
Figure l l . Extracted ion electropherograms of the 10 major toxins in the venom of N. melanoleuca observed during full-scan CEIESI-MS (16-ng on-column Injection of the total venom): peak 8, m/z = 1669, phospholipases DE-I11 and DE-IIIA; peak 9, m/z = 1664.5, M, = 13 308.3; peak 10, m/z = 1672, M, a k = 13 366.6; peak 11, m/z = 1424.5, h.4, cdk= 71 18.8; peak 12, m/z = 1110, toxin b; peak 13, m/z= 1114,h.4,,I,=7792.0;peak 14,m/z= 1120,M,ak=7832.6; peak 15, m/z = 1493, M, a k = 13 430.9; peak 16, m/z = 972, toxin d; peak 17, m/z = 1113, toxin Viil.
DE I11 and DE-IIIA42and toxin Vii141 are readily separated with migration times of 7 and 9 min, respectively. The venom of N . melanoleuca has not been quantified, but from previous e x p e r i e n ~ e , 'these ~ , ~ ~traces result from the injection of tens to hundreds of femtomoles per toxin. At this level, more typical CE peaks with separation efficiencies in the order of 20 000230 000 are obtained. Full-scan spectra can still be obtained Analytical Chemistry, Vol. 66, No. 18, September 15, 1994
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Table 1. Identification of Peptides Noted in the Venom of Na/a melanoleuca during Full-Scan CE/ESI-MS Experiments. injection mol wt peak no.
m/z
1 2 3 4 5 6 7 8
1667.5 1506 1683 1268 1130 1061.5 1141.5 1669.5
9 10 11 12 13 14 15 16 17 18
1664.5 1672 1424.5 1110 1114 1120 1493 972 1113 1144
charge states noted 60 ng 16 ng migration time (min) column efficiency
+ +
+8, +7 +6, +5 +6, +5
+ +
-
+ + + + + + + + + + +
+ + + + + + + + + +
+ +
+7, +6, + 5 +9, +8, +7 +8, +7 +9, +8 +6, +5, +4 +7, +6 +8, +7, +6 +7, +6 +9, +8, +7 +7, +6, +5 +7, +6, +5 +7, +6, +5
-
+
6:08* 6:08* 6:13* 6:40* 7:55* 8:07* 9:23* 6:48
-
6:53 6:53 7:16 7:38 7:53 7:53 8:30 8:33 8:43 9:49*
-
17 760* 39 200* 30 830* I 4 570* 34 720* 52 560* 55 400* 87 310 129 620 20 740 47 890 129 120 137 720 117 345 230 550 119 020 112 510
exptl
theor
accuracy (%)
toxin (ref)
0.031 0.014 0.009
Vii2 (40) Ph. DE-IIIA (42) Ph. DE-I11 (42)
0.005
b (39)
0.011
d (39) Viil (41) Vii3 (40)
13453.0 6336.0 6769.4 6841.1 6843.2 13348.9 13350.9 13345.7 13346.9 13308.3 13366.6 7118.8 7761.6 7762 7792.0 7832.6 13430.9 6794.9 6795.7 6673.5 6674.1 6857.5 6856.3
0.009 0.018
4 Peptides are referred to using a single multiple charge state (usually the most abundant). Migration times and column efficiencies marked with an asterisk refer to Figure 7.
E n q
c
o
600
800
1000
1200
14V
I600
1x00
200n
Figure 12. On-line spectra obtained from full-scan CE/ESI-MS of N. melanoleuca venom as shown in Figure 10: (A) toxin of molecular mass 71 18.8 Da; (B)toxin d.
at this level (Figure 12) though the spectral quality means that the spectra are less conclusive than at the higher injection level. An additional gain using a magnetic sector instrument vs the quadrupole was that the additional resolution aided accurate molecular mass assignment of peptides. Using CEESI-SIM-MS on a quadrupole instrument, detection of peptides was enhanced by decreasing the instrumental resolution. This meant that the each mass peak could be up to four mass units wide. During CE-ESI-SIM-MS of thevenom from N . melanoleuca, several species were noted in the electropherogram for m/z = 1113 such that these peaks could relate to peptides with multiple charge states from m/z = 1111 to m/z = 11 15.46 Mass assignment in these experiments relied on the provision of at least two charge states, together with a common migration time. The sector instrument was able (46) Perkins, J . R. unpublished data
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Analytical Chemistry, Vol. 66, No. 18, September 15, 1994
to discriminate between one peptide with a charge state at m/z = 1113.4 and another peptide with one at m/z = 1113.8. These peptides are readily separated using CE, and their extracted spectra provided accurate molecular masses of 6673.5 and 7792.0 Da, respectively. The toxins detected during CE/ESI-MS experiments are summarized in Table 1. The mass accuracies obtained during CE/MS experiments for the previously sequenced toxins range from 0.005% (toxin b) to 0.031% (toxin Vii2).
CONCLUSION Capillary electrophoresis has been successfully interfaced with electrospray mass spectrometry on a high-performance magnetic sector instrument. The interface is capable of accommodating capillary electrophoresis with either positive or negative polarity, as demonstrated by standard peptide mixtures and a snake venom. All experiments were performed in the full-scan mode so that useful spectra could be obtained for analytesunder observation. The snakevenom was analyzed using two sample injection volumes, where a larger injection supplied useful on-line spectra together with degraded electrophoretic peaks while the smaller injection volume yielded sharper electrophoretic peaks. CE/ESI-MS on a sector instrument provided additional resolution for superior mass assignment of multiple charged species of similar mass to charge ratios compared to quadrupole instrumentation. Received for review February 28, 1994. Accepted June 2,1994." *Abstract published in Adounce ACS Abstrocts, July 15, 1994.
