1058
Anal. Chem. 1991, 63, 1658-1660
significant improvements in detection limita result from use of a sparse array of many small electrodes.
ACKNOWLEDGMENT We are grateful to Neil Danielson of Miami University, Oxford, OH, for a generous gift of sized Kel-F particles.
LITERATURE CITED (1) Andenon, J. L.; CheSnOy, D. J. Anal. Chem. 1080, 52, 2158. (2) M y , D. J.: AndUUOn, J. L.; WeQshear, D. E.; Tallman, D. E. Anal. chkn.A& 1081, 124, 321. (3) WeiEshaar, D. E.;Telhnan. D. E.; Anderson, J. L. Anal. Chem. 1081, 53, 1809. (4) Caudll, W. L.; Howell, J. 0.;Wlghtman. R. M. Anal. Chem. 1082. 54, 2532. (5) Tallman, D. E.; Wdsshaar, D. E. J . Llq. CYmnatogr. 1083, 6, 2157. (8) Webrshrrar, D. E.; Talhnan, D. E. Anal. Chem. 1083, 55, 1148. (7) Anderson, J. L.; Whiten, K. K.: Brewster, J. L.; Ou, T. Y.; Nonidez, W. K. AMI. CWm. 1085, 57, 1368. (8) Foedldc, L. E.; Anderson, J. L. Anal. Chem. 1088, 58, 2481. (9) FOSdICk, L. E.; Anderson, J. L.; Baglnskl. T. A,; Jaeger, R. C. Anal. Chem. 1086, 58, 2750. (10) bbl, F.; Anderson, J. L. AM@t 1086, 110. 1493. aw,L. J., Jr.; Ostscyoung, J. Anal. Chem. 1000. 62, 2625. (11) M (12) FWlnovsky, V. Yu. Ebcbodnkn. Acta 1080. 25, 309. (13) Moldoveanu, S.: Anderson, J. L. J . Electroanal. Chem. Intetfacial Electrochem. 1086, 185, 239. (14) COfm,D. K.; Tallman, D. E. J . Electroanal. Chem. InterfacialElecb.0chem. 1086, 188, 21.
(15) Cope, D. K.; Tallman. D. E. J . Ebetrmnal. C h m . IntetfadpIElecb.0&em. 1086, 205, 101. (IS) Weber, S. 0. Anal. Chem. 1080, 61. 205. (17) Lankelma, J.; Poppe,H. J . clwometogr. 1076, 125, 375. (18) Morgan, D. M.; Weber, S. 0. Anal. Chem. 1084, 56, 2580. (19) Anderson, J. L.; OU. T. Y.; Mddoveanu, S. J . EkwIhJanal. Chem. Interfacial Electrochem. 1085, 196, 213. (20) Ou,T. Y.; Moldoveanu, S.; Anderson, J. L. J . Ehtroenal. Chem. Interfadsl E k t r m . 1088, 247. 1. (21) Prabhu, S.; Anderson, J. L. Anal. Chem. 1087. 50, 157. (22) Ou. T. Y.; Anderson, J. L. J . Electroanel. Chem. Interfacial Electrochem. 1091. 302, 1. (23) Foley. J. P.; Dorsey, J. 0. Anal. Chem. 1083, 55, 730. (24) Meschl, P. L.; Johnson, D. C. Anal. Chlm. Acta 1981, 124, 303. (25) Anderson, J. E.; Tallman, D. E.; Chesney, D. J.; Anderson, J. L. Anal. Chem. 1978, 50, 1051. (26) Amatore. C.: Saveant, J. M.; Tessier, D. J . EkwIhJanel. Chem. Interfads1E l e c t ” . 1083, 147, 39. (27) Petersen, S. L.; Welsshaar, D. E.; Tallman, D. E.; Schulze. R. K.; Evans, J. F.; Desjarlais, S. E.; Engstrom, R. C. Anal. Chem. 1988, 60, 2385. (28) m i , T.; Tokuda, K.; Matsuda. H. J . Electroenal. Chem. Interfecial Electrochem. 1078, 80. 247. (29) Aokl, K.; Tokuda, K.; Matsuda, H. J . Electroanel. Chem. Interfacial Elect”. 1087, 217, 33. (30) Petersen, S. L.; Tallman, D. E. Anal. Chem. 1000, 62, 450. (31) Anderson, J. E.; Montpomery. J. B.; Yee, R. Anal. Chem. 1000. 83, 653.
RECEIVED for review February 15,1991. Accepted May 13, 1991.
TECHNICAL NOTES Comblnatlon Eiectrospray-Liquid Secondary Ion Mass Spectrometry Ion Source Damon I. Papac, Kevin L. Schey, and Daniel R. Knapp* Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425
INTRODUCTION Recent developments in electrospray ionization (I,2) have prompted widespread interest among mass spectrometrista studying large molecules. Most of these investigators use, and will continue to use, liquid secondary ion mass spectrometry (SIMS) ionization in their work. In many laboratories the same mass spectrometer will be used for both electrospray and liquid SIMS work, necessitating reconfiguration of the instrument for changing between the two ionization methods. Faced with this prospect, we decided to implement electrospray ionization on our triple-quadrupole instrument (Nermag R30-10)in a manner that would allow easy switching between liquid SIMS and electmpray modes. This note describes the successful implementation of a combination electrosprayliquid SIMS ion source on a quadrupole type mass spectrometer. We had previously modified our instrument to incorporate a cesium ion gun (3)for liquid SIMS analysis of peptides. The cesium ion gun was mounted on the ion source with the cesium ion beam coaxial with the sample probe and perpendicular to the quadrupole axis. To implement electrospray ionization, we chose the recently published design of Chowdhury, Katta, and Chait (4), which employs a heated metal capillary for desolvation. The repeller on the standard Nermag FAB ion source was replaced by a hemispherical electrode attached to the end of the electrospray source. The resulting device gave comparable performance in liquid SIMS mode to the previous configuration and allowed rapid changeover (30min) between liquid SIMS and electrospray modes within the time required 0003-2700/91/0383-1858$02.50/0
to heat and cool the ion source pellet in the cesium ion gun. This combination ion source permits the use of both modes of ionization without the need to reconfigure the instrument. EXPERIMENTAL SECTION A diagram of the ion source is shown in Figure 1. The components are as follows: (A) Hamilton (Reno, NV) 1705RN syringe with Hamilton 80426 needle (25 gauge) driven by a Harvard Apparatus (South Natick, MA) Model 11 syringe pump (the high voltage required to generate the spray is applied to the syringe needle; a Teflon insulating cap is placed over the syringe plunger handle to prevent discharge through the syringe plunger to the pump); (B) stainless steel counterelectrode disk insulated from the stainless steel capillary with Kel-F bushing; (C) Upchurch Scientific (OakHarbor, W A ) P-640Kel-F adapter, 1/4-28thread to 10-32Fingertight chromatography fitting sealed to flange with Teflon O-ring; (D)20 cm X 0.062 in. o.d., 0.020 in. (0.50mm) i.d. 316 stainless steel tubing (Upchurch Scientific U-103); not shown is heater wire (0.020 in. Nichrome) in fiberglass sleeving wrapped on capillary and an iron-constantan thermocouple to monitor capillary temperature; (E) Kel-F bushing to align capillary end with skimmer (the bushing is mounted in a stainless steel plate mounted on two threaded standoffs); (F) skimmer cone with 0.5-mm orifice [Vestec (Houston, TX) VT 1020Al located ca. 3 mm from end of capillary; (G) hemispherical electrode (radius 0.375 in.; center of radius at entrance of f i t ion source lens) which serve8 as the repeller for liquid SIMS operation [the skimmer cone and hemispherical electrode are electrically connected to the ion source repeller voltage supply (0-40 V)];(H) Teflon washer (0.030 in.) which insulates the skimmer mounting plate from the housing (the mounting plate is attached with six 2-56Nylon screws);(I) standard lenses of the Nermag R30-10FAB ion source; (J) first 0 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL.
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Flgure 1. Dlaqam of the comblnatbn electrospray-liquid SIMS ion source mounted In the Nermag R30-10 tandem mass spectrometer. For explanation of the labeled components, refer to the Experimental Section.
4W
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Figure 3. Eleclrospray ionization mass spectrum of equine myogkbln.
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Flgurr 2. Electrospray ionization mass spectrum of bovine insulin.
quadrupole masa analyzer; (K)cesium ion gun (Antek, Palo Alto, CA) mounted to FAB ion source block; (L) Nermag FAB probe (shown retracted from ion source); (M) Upchurch ScientificFlOO Kel-F Fingertight chromatography fitting used with 18-gauge copper wire in in. 0.d. Teflon tubing to provide electrical feedthroughs for heater and thermocouple wires (one shown of four); (N) Conflat type flanges (MDC Vacuum Products Corp., Hayward, CA), 2.75 in. o.d., mounted with a Viton O-ring in place Conflat type of copper gasket to maintain constant spacing; (0) flanges, 4.5 in. o.d., mounted with a Viton O-ring; (P)pumping port connected via butterfly valve to cold finger and mechanical vacuum pumps (three Alcatel Model 2012A pumps, each 310 L/min in parallel). For electrosprayanalysis the samples were dissolved in 47:47:6 (v/v/v) water/methanol/acetic acid and delivered to the needle at a rate of 0.5-1.0 rL/min. Concentrations of approximately 50 pmol/pL were used with no attempt in this work to maximize sensitivity. Typical operating voltages were as follows: needle, 4.4 kV; capillary and counterelectrode plate, 250 V; skimmer/ repeller, 30 V. The capillary was maintained at a temperature of 90-100 OC. For liquid SIMS analysis the samples were applied in 5% acetic acid or 0.1% trifluoroaceticacid to a f i i of thioglycerol onto the end of a gold-plated 2 mm diameter stainless steel probe tip. Cesium ions (7 kV energy) were used to desorb sample ions. Data were acquired by using the Nermag (Delsi Nermag, Houston, TX) Sidar data system in the MS/MS mode with nominal m w data acquired in the peak-stepping mode. The mass scale was calibrated by using cesium iodide in liquid SIMS mode, and the calibration was used for both liquid SIMS and electrospray data acquisition. Molecular weights were calculated by multipying the observed m/z by the charge state and expressed as the mean standard deviation of the values determined for each charge State.
The electrospray ion source has also been tested on a custombuilt tandem masa spectrometer based upon Extrel (State College,
miz
Figure 4. Electrospray lonizatlon mass spectrum of bovine serum albumin.
Minor Components
Pd a 4 -
4w
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Figure 5. Electrospray ionization mass spectrum of porpoise (phocoenoMes dalli) relaxin.
PA) quadrupoles (5). The geometry of this instrument required increasing the distance between the skimmer F and flanges N as well as lengthening the capillary D. This was accomplished by addition of an 8-cm extension tube onto which the skimmer assembly was mounted. The capillarywas extended by addition of a 10-cm length connected with a zero dead volume union. Data were collected on this instrument with a Teknivent (St. Louis, MO) Vector Two data system with data acquired in nominal mass mode. Electrospray performance was comparableto that obtained on the Nermag instrument, indicating that the length of the capillary is not critical (as also observed by Chait et al. (6)).Due to the geometry of this instrument (probe located 90° with respect to the FAB gun) it was not possible to operate in the FAB mode
Anal. Chem. 1991, 63,1660-1664
1660
with the electrospray source installed. The protein samples used to produce the spectra in Figures 2-4 were obtained from Sigma Chemical Co. (St. Louis,MO) and used without further purification. The sample used for the data shown in Figure 5 was provided by Dr.Christian Schwabe of the Medical University of South Carolina.
mass differences of the components could be used to gain information on terminal amino acids (relaxin consists of two chains connected by disulfide bonds). For example, the difference of 71 mass units between components I and II would suggest a terminal alanine.
RESULTS AND DISCUSSION
CONCLUSIONS A combination electrospray-liquid SIMS ion source has been constructed that allows use of both modes of ionization without the need for physical reconfiguration of the instrument. The electrospray data obtained on samples of bovine insulin (mol wt 5733.6; error 0.034%),equine myoglobin (mol wt 16950.5; error 0.037%), and bovine serum albumin (mol wt 66267; error 0.4%) are comparable to previously published data (4, 7). The electrospray source also performed satisfactorily on a second tandem quadrupole instrument which required a longer desolvation capillary, indicating that the design should be adaptable to a variety of instruments.
The combination ion source gave comparable performance in liquid SIMS mode to the standard Nermag FAB ion source (data not shown), indicating that the hemispherical repeller was an adequate substitute for the curved metal strip repeller on the standard FAB ion source. The results of sample analysis in electrospray mode are shown in Figurea 2-5. Figure 2 shows data obtained from analysis of bovine insulin. The measured molecular weight (5731.7 f 1.5) was within 0.034% of the calculated value (5733.6). Figure 3 shows results obtained for equine myoglobin; the measured molecular weight (16 944 f 5) was within 0.037% of the calculated molecular weight (16950.5). The accuracy of these measurements is limited by the acquisition of nominal mass data and could likely be improved by acquisition of “profile”data and measurement of mass to charge ratios to tenths of a unit. Figure 4 shows data obtained on bovine serum albumin, the largest protein examined with this ion source. The measured molecular weight (66541 f 39) was within 0.4% of the calculated weight (66 267). The difference between the measured and calculated masses for this protein are comparable to those reported by other investigators (4, 7) and could be due to bound ions (e.g. Ca2+)other than protons and/or modifications of the protein. Figure 5 shows data obtained on a sample of porpoise relaxin (a protein structurally similar to insulin) for which only a partial sequence was known from Edman degradation. The sample spectrum contained one major and several minor series of peaks. Manual calculations on the mixture data (assuming molecular weight for relaxin in the 5000-6000 range) permitted identification of a major peak series (with charge states of +4 to +7) of molecular weight 6057.9. Components of masses 5986.4 (+3 to +7), 5883 (+4 to +6), 5726 (+3 to +6), 5629 (+5, +6), 5478 (+6; charge assignment based upon assumption that this peak was from a relaxin related molecule), and 5395 (+5, +6) were also observed. If these multiple components were the result of proteolytic trimming occurring in the isolation process, the
ACKNOWLEDGMENT We thank S. K. Chowdhury and B. T. Chait for helpful discussion of their electrospray design. Special thanks go to Clifton Harvey for his expert machine work in construction of the ion source. We thank Vicki Wysocki, Virginia Commonwealth University, Richmond, VA, for the opportunity to test the ion source on her instrument.
LITERATURE CITED (1) Whltehouse, C. M.; Dreyer, R. N.; Yamashlta. M.; Fenn, J. B. Ana/. chem.1985, 57, 875-879. (2) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whbhouse, C. M. SdenCe 1989, 246, 84-71. (3) Aberth, W.; Straub. K. M.; Burlingame, A. L. Anal. Chem. 1982, 54, 2029-2034. (4) Chowdhuy, S. K.; Katta, V.; Chalt, B. T. Repid Commun. Mass Specbpm. 1990, 4 , 81-87. ( 5 ) Callahan, J. H.; King, F. L.; Ross, M. M.; Wysodtl, V. H. Roc. Annu. coni. Am. Soc. Mass Speck” 1990, 38, 898-899. (8) Chalt, B. T. Presented at the ASMS Fall Workshop on Ebctrospray Ionization, Nov. 5-8. 1990, Chicego, IL. (7) Baaynskl, L.; Bronson. 0. E. Repld Commun. Mass spectrom.1890, 4 , 533-535.
RECEIVED for review February 6, 1991. Accepted April 15, 1991. This work was supported in part by NIH Grant EY08239. This work was presented at the ASMS Fall Workshop on Electrospray Ionization, Nov 5-6, 1990, Chicago, IL.
Method for the Eiectrospray Ionization of Highly Conductive Aqueous Solutions Swapan K. Chowdhury and Brian T. Chait* Laboratory of Mass Spectrometry, The Rockefeller University, 1230 York Avenue, New York, New York 10021
INTRODUCTION The electrospray phenomenon (also known as electrohydrodynamic atomization) is a proc of disintegration of a liquid surface in the presence of a s rong electric field into a spray of f i e , highly charged droplets. A number of studies have been carried out, going back more than 70 years, aimed at gaining an understanding of the fascinating physical processes governing the spray (1-3). Considerable interest in the electrospray process has also arisen because it has found wide applications for such diverse purposes as electrostatic emulsification (2e, 4, electrostatic painting (5), paint spraying (2e, 6), fuel atomization in combustion systems (7),crop spraying (2e, 8), and a method for sample preparation for 0003-2700/9 110363-1680502.50/0
@-countingexperiments (9) and %%f plasma desorption mass spectrometry (10). The most recent resurgence of interest in electrospray has arisen in connection with the technique of electrospray ionization mass spectrometry (11-1 7). In this technique, solutions of involatile organic molecules and biopolymers (such as proteins and DNA) are electrosprayed at atmospheric pressure to produce a large number of small highly charged droplets containing the component(s) of interest. The solvents are rapidly evaporated from the droplets, and the residual biopolymer ions are transported through differentially pumped orifices or capillaries into a mass spectrometer where the mass-tu-charge ratio (m/z)values of the ions are accurately determined. By using electrospray 0 I991 American chem(cal Society
