Anal. Chem.
3528
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65.3528-3533
Factors Affecting Electrospray Ionization of Effluents Containing Trlfluoroacetic Acid for High-Performance Liquid Chromatography/Mass Spectrometry J a m s h i d Eshraghi and S w a p a n
K.Chowdhury’
Analytical Science Department, Sterling Winthrop Inc., 9 Great Valley Parkway, Malvern, Pennsylvania 19355 INTRODUCTION Electrospray ionization ( E S P ) ,ion spray (pneumatically assisted electrospraybfi),and matrix-misted laser desorption74 techniques have emerged as powerful methods for the characterization of large, nonvolatile, and labile molecules. The techniques, in particular, have made significant impact in biochemistry and biotechnology.zsJoJ1 The molecular weights of peptides and proteins can now be determined to an accuracy of better than 0.01 96 up to 20 OOO and at lower accuracy for higher molecular weights. The accuracy obtained from these methods is much higher than that obtained using a sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis rnethod.lzJ3 In addition, the amount of sample required to perform such measurements has been reduced to the subpicomole level. Although an accurate molecular weight determination provides confirmation of the primary structure of a peptide or a protein, detailed structural identification (e.g., sequence) or determination of the location of modifications and mutation sites cannot be made from molecular weight measurement alone. For structural elucidation, peptides and proteins are chemically or enzymatically cleaved into smaller fragments and the molecular weight and the structure of the resulting fragments are determined by a combination of techniques, such as amino acid analysis, Edman sequencing, mass spectrometry, and tandem spectrometry (MS/MS). The characterization of complex mixtures of a large number of peptides generated by chemical or enzymatic digestion is difficult by mass spectrometry without prior separation into groups containing smaller number of components. Direct extraction from plant or animal sources may also generate complex mixtures of materials which are difficult for direct mass spectrometric characterization.
High-performance liquid chromatography coupled with electrospray or ion spray mass spectrometry (LC/MS) provides an excellent combination of techinques to obtain an on-line separation together with molecular weight and structural determination of separated components. LC/MS with ESI provides specificity and selectivity, high sensitivity, and molecular characterization, all of which cannot be obtained from either LC or ESI-MS alone. Fenn and co-workers’ were the first to demonstrate the possibility of performingLC/MS using an electrospray ionization source. Henion and coworkers subsequetnly published extensive research on LC/ MS and LC/MS/MS using an ion spray device.bJ41g These investigators have demonstrated that the use of pneumatic nebulization allows electrospray ionization of LC effluents at a flow rate of up to 40 pL/min”Jk16J8 and up to 500 pL/min by additionally heating the effluents.17* By placing a liquid shield between the sprayer and the ion sampling orifice, they were able to perform ion spray LC/MS with a flow rate of up to 2 mL/min.I7b Using LC/MS and LC/MS/MS, they examined the enzymaticdigests of ~-lactoglobuliis,14bbovine cytochrome c,14bbovine albumin,lBand glycopeptides.18 Carr and co-workers,rn using an ion spray LC/MS/MS system,have investigated a variety of glycopeptide models and glycoprotein digests of a soluble complementreceptor type I,a glycoprotein of mass 240 OOO u.ab In addition, there are a number of publications that describe the use of the ion spray LC/MS technique for the characterization of small molecules and p e p t i d e ~ . ~ lAl-l~these ~ studiesklku were performed on ion spray sources or prototypes developed by Henion and coworker~.~* In contrast, there are only a small number of LC/MS investigations described in the literature performed on other electrospray sources. Hunt et al.26*28have utilized microcapillary ESI LC/MS and ESI LC/MS/MS to perform sequence analysis of a large number of different peptides and
* Author to whom correspondence should be addressed.
(14)(a) Covey, T. R.; Lee, E. D.;Bruins, A. P.; Henion, J. D.Anal. (1)(a) Yamaahita, M.;Fenn, J. B. J.Phys. Chem. 1984,88,4451-4459. Chem. 1991,63,732-739. (b) Huang, E.C.; Henion, J. D. J. Am. Soe. (b) Yamsshita, M.; Fenn, J. B. J. Phys. Chem. 1984,88,4671-4675.(c) Mass Spectrom. 1989,1, 158-165. Whitehouse,C. M.; Dreyer,R. N.; Yamaahita, M.; Fenn, J. B.Ana1. Chem. (15)Huang, E. C.; Wachs, T.; Conboy, J. J.; Henion, J. D. Anal. Chem. 1985,57,675-679. 1990,6,713A-725A. (2)Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F. Mass Spectrom. Rev. 1990,9,37-70. . (16)Huang, E. C.; Henion, J. D. Anal. Chem. 1991,63,732-739. (3)(a)Smith,R.D.;Loo,J.A.;Edmonds,C.G.;Barinaga,C.J.;Udeseth, (17)(a) Lee, E. D.;Henion, J. D. Rapid Commun.M u s Spectrom. 1992,6,727-733. (b) Hopfgartner, G.; Wachs, T.; Bean, K.; Henion, J. H.R.Anal.Chem.1990.62.882-889.(b)Smith,R.D.:Loo,J.A.:Edmonds, D. Anal. Chem. 1993,65,439-46. C. G.; Udeseth, H. R. Idass Spectrom. Rev. 1991,10;359-451..(c) Smith, (18)Conboy, J. J.; Henion, J. D. J.Am. Soc. Mass Spectrom. 1992,3, R. D.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1988,60,1948-1952. 804-814. (4)(a) Chowdhury, S. K.; Katta, V.; Chait, B. T. Rapid Commun. (19)McLucky,S.A.;Berkel,G.J.V.;Glish,G.L.;Huang,E.C.;Henion, Mass Spectrom. 1990,4,81-87.(b) Chowdhury, S. K.; Katta, V.; Chait, J. D. Anal. Chem. 1991,63,375-383. B. T. NATO ASI Series; Standing, K. G., Ens, W., Eds.; Plenum Press: (20)(a)Hemling,M.E.;Roberta,G.D.;Johnson,W.;Carr,S.A.Biomed. New York, 1991;pp 201-210. Enuiron. Mass Spectrom. 1990,19,677-691. (b) Huddleston, M.J.; Bean, (5)(a) Bruins, A. P.; Covey,T. R.; Henion, J. D. Anal. Chem. 1987,59, M. F.; Carr, S. A. Anal. Chem. 1998, 65, 877-8134, (c) Carr, S. A.; 2642-2646. (b) Covey, T.R.; Bonner, R. F.; Shushan, B. I.; Henion, J. D. Rapid Commun. Mass Spectrom. 1988,2,249-256. Huddleston, M. J.; Bean, M. R. Protein Sci. 1993,2,183-196. (21)(a) Pleasance, S.; Blay, P.; Quilliam, M. A.; O’Hara, G. J. (6)Feng,R.;Konishi,Y.;BeU, A.W. J. Am. SOC. Mass Spectrom. 1991, Chromatogr. 1991,558,155-73.(b) Pleasance, S.;Quilliam,M.A.; Marr, 2,387-401. J. C. Rapid Commun. Mass Spectrom. 1992,6,121-127.(c) Pleaeance, (7)(a) Karas, M.; Hillenkamp, F. Anal. Chem. 1988,60,2299-2301.(b) S.; Kelly, J.: LeBlanc, M. D.: Quilliam, M. A.; Boyd, R. K. Biol. Mass Karas, M.; Backmann, D.; Bahr, U.; Hillenkamp, F. Int. J.Mass Spectrom. Spectro-h. 1992,21,675-687; Ion Processes 1987, 78,53-68. (22)Wandverson. D.: Arnold. M. E.: Jemal,. M.:. Cohen, A. I. Biol. (8)Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Mass Spectrom. 1992,21,189-94. Rapid Commun. Mass Spectrom. 1988,2,151-153. (23)Suzuki-Sawada,J.; Umeda,Y.; Kondo, A.;Kato, I. Anal. Biochem. (9)(a)Beavis, R. C.; Chait, B. T. Anal. Chem. 1990,62,1836-1840. (b) 1992,207,203-207. Beavis, R. C.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1990,87,6873(24)Hogge,L.R.;Abrama,G.D.;Abrams,S.R.;Thibault,R.;Pleasance, 6877. (10)Carr, S.A.; Hemling, M. E.; Bean, M. F.; Roberta, G. D. Anal. S. J. Chromatogr. 1992,623,255-63. (25)Hunt, D. F.;Alexander, J. E.; McCormick, L. A.;Martino, P. A.; Chem. 1991,63,2802-2824. Michel, H.; Shabanowitz, J.; Sherman, N.; Moseley, M. A.;Jorgensen, J. (11)Chait, B. T.;Kent, S. B. H. Science 1992,257,1885. W.; Tomer, K. B. In Techniques in Protein Chemistry; Villafranca, J. J., (12)Weber, K.; Osborn, M. J.Biol. Chem. 1969,224,4406-4412. Ed.; Academic Press: New York, 1991;Vol. 11. (13)Segrest, J. P.; Jackson, R. L. Methods Enzymol. 1972,28,54-63. 0003-2700/93/0365-3528$04.00/0
0 1993 American Chemlcal Soclety
ANALYTICAL CHEMISTRY, VOL. 65, NO. 23, DECEMBER 1, 1993
proteins. Using this technique, these authors have demonstrated the fractionation and determination of the sequences of subpicomolar amounts of peptides isolated from the major histocompatibility complex molecules.26 Some applications of electrospray LC/MS were also reported by Lee and coworkers,m Griffin et al.," Caprioli and co-workers,m and Voyksner et al.30 These investigationswere done on electrospray sources developed by Fenn and co-workers1*2and prototypes. In the present paper, we report an investigation to optimize the performance of an electrospray ion source1~~28 on a TSQ mass spectrometer to obtain high sensitivity and signal stability for the ionization of LC effluents with mobile phases containing aqueous 0.1 9% TFA. TFA is known to be one of the best YmOdifiersnfor liquid chromatography and does not interfere with the spectrophotometric detection of the effluents in the ultraviolet regions. As a result, biologists use mobile phases containing 0.1% TFA for LC. In order to maintain chromatographic profiles similar to those obtained by biologists and biochemists (for an unambiguous identification of LC peaks), we are often required to use similar mobile phases. It is known that TFA, being a strong acid, increases the conductivity of water, which has high surface tension. Electrospray of solutions with high surface tension and high conductivity becomes difficult",313" as a result of the instability (31,321 of the "Taylor from the tip of which highly charged droplets are emitted at a steady rate for a good electrospray. The instability causes frequent breakdown of the spray and reduction in the analyte ion signal.3334~3638The present report describes our efforts to overcome the problem. All the previous investigations that report the LC/MS of mobile phases containing up to 0.15% TFA performed on ion spray sources used a minimum organic content of 5%.15J'3 Griffin et al." reported the use of ESI LC/MS with a capillary column using a gradient of acetonitrile (0.085% TFA) and water (0.1% TFA) at a flow rate of 2 pL/min. We demonstrate here electrospray ionization of biomolecules in aqueous 0.15% TFA on an Analytica (Analytica at flow rates up to 30 of Branford, CT) ESI source1*~~"*39 pL/min and LC/MS of effluents containing aqueous 0.1% TFA with high sensitivity.
EXPERIMENTAL SECTION Materials. Ammonium bicarbonate, ammonium hydroxide, trifluoroaceticacid (HPLC grade),and acetonitrile (HPLCgrade) (26) (a) Hunt, D. F.; Michel, H.; Dickinson, T. A.; Shabanowitz, J.; Cox, A. L.; Sakaguchi, K.; Appella, E.; Grey, H. M.; Sette, A. Science 1992,256,1817-1820. (b) Hunt, D. F.; Henderson, R. A.; Shabanowitz, J.; Sakaguchi,K.; Michel,H.; Sevilir,N.; Cox, A. L.; Apella,E.; Engelhard, V. H. Science 1992,255,1261-1266. (27) (a) Davis, M. T.; Lee, T. D. Protein Sci. 1992,1, 935-944. (b) Swiderek, K. M.; Chen, S.; Feiatner, G. J.; Shively, J. E.; Lee, T. D. In Techniques in Protein Chemistry; 3, Angeletti, R. H., Ed.; Academic Press: San Diego, CA, 1992; Vol. 3, pp 457-465. (28) Griffin, P. R.; Coffman, J. A.; Hood, L. E.; Yates, J. R., I11 Znt. J. Mass Spectrom. Zon Processes 1991,111, 131-149. (29) Suter, M. J.-F.; Dague, B. B.; Moore, W. T.; Lm,S.-N.; Caprioli, R. M. J. Chromatogr. 1991,563,101-116. (30) (a) Straub, R.; Voyksner, R. D.; Keever, J. T. J. Chromtogr. 1992,627,173-86. (b) Lin, H.-Y.; Voyksner, R. D. Anal. Chem. 1993,65,
451-456. (31) (a) Hayati, I.; Bailey, A. I.; Tadros, Th. F. Nature 1986,319,41. (b) Zbid. J. Colloid Interface Sei. 1987,117,205-221. (c) Zbid. 222-230. (32) Smith, D. P. H. ZEEE Trans.Znd. Appl. 1986,ZA-22,527-535. (33) Chowdhury, S. K.; Chait, B. T. Anal. Chem. 1991,63,1660-1664. Mass (34) Ikonomou, M. G.; Blades, A. T.; Kebarle, P. J. Am. SOC. Spectrom. 1991,2,497-505. London 1964,280, 383. (35) Taylor, J. Proc. R. SOC. Mass (36) Wampler, F. M.; Blades, A. T.; Kebarle, P. J. Am. SOC. Spectrorn. 1993,4, 289-295. (37) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989,246,64-71. (38) Fernandez de la Mora, J.; Roseel-Llompart,J. Proc. 39th ASMS Conf. Mass Spectrom. Allied Topics, Nashville, TN, May 1991; pp 441442.
BS2Q
were obtained from Baker Analyzed; 2-methoxyethanol (glass distilled) was obtained from Aldrich; and distilled water was prepared with a Milli-Q distillation apparatus from Millipore System. Porcine 8-malenocyte stimulating hormone (M2018, DEGPYKMEHFRWGSPPKD), ACTH (1-24) (A0298, SYSMEHFRWGKPVGKKRRPVKVYP), angiotensin I1 (A9525, DRVYIHPF), bombesin (B5508, pGluQRLGNQWAVGHLMNHz), des-Tyrl-leucine enkephalin (E7255, GGFL), bradykinin (B3259,RPPGFSPFR), horse heart cytochrome c (C2506), and bovine heart cytochrome c (C2037) were obtained from Sigma ChemicalCo. Renin substrate (M-1115,DRVYIHPFHLLVYS, horse sequence) was purchased from Bachem Bioscinece Inc., Philadelphia, PA, and trypsin (1047 841) from Boehringer Mannheim GmbH, Mannheim, Germany. Digestion with Trypsin. A 96-pg sample of bovine cytochrome c was added to a 8 pmol/pL trypsin solution prepared in 0.06 M NH4HC03 solution (pH = 8.6) to obtain an enzymeto substrate ratio of 1:25. Horae cytochromec was digestedsimilarly in 5 pmol/pL solution of trypsin at the same pH and enzyme to substrate ratio. The bovine protein was incubated for 20 h and that from horse for 30 h at 37 OC in a constant-temperature heating unit (Thermomixer, Eppendorf). The digestions of the two proteins were carried out on separate occasions and the difference in digestion time was an effect of the different concentrations of trypsin used. The digestedmaterials werespindried under vacuum using a Speed Vac concentrator and dissolved in 0.1 % TFA to prepare a 20 pM solution. Only 10 pmol was used for LC/MS through a 0.5-pL loop injector system. High-Performance Liquid Chromatography. Separation of control peptide mixtures and those obtained from proteolytically digested proteins were obtained by reversed-phasecapillary HPLC with several gradients (discussed later) using a Waters 600 delivery apparatus controlled by a Waters 600 MS system. The HPLC pump was operated at flow rates between 300 and 600 pL/min prior to splitting the liquid flow with a ratio of 1:100 usinga commercialsplitter (Acurate,LC packings). The smaller fraction passed through a 0.5-pL injection loop system followed by a microcapillary HPLC column (Fuscia from LC packings; dimension, 320 pm i.d. X 15 cm; static phase, Vydac C-18, 300 A). Effluents that exit the capillary column were monitored by a Spectroflow 783 UV detector set at an wavelength of 214 nm. The UV detector was connected with the DECstation 5000/120 of the Finnigan MAT TSQ-700 mass spectrometer (San Jose, CA). The setup is similar to that described by Griffii et a1.m The solutions exiting the detector were fed into the electrospray ion source1@through a 25-pm fused-silica capillarytube. The sample solution (for infusion experiments) and the sheath liquids were delivered from hypodermic syringes (100 and 500 pL) using Harvard apparatus (Model 22) and Sage Instrument (Model 341B) syringe pumps. The mobile phases were A = 0.1% aqueous TFA and B = 0.1 % TFA in acetonitrile. For both the control peptide mixtures and the tryptic digests of cytochromes c, a flow rate of 4 pL/min after splitting was used for electrospray ionization. For the peptide mixtures, the change of percent of A with time was as follows (t in min, % A): 0,100,3,100; 15,80; 60,60; 80,20; 9 0 , l O ; and 100, 100. For the digests: 0,100; 5,100; 30,55; 120,30; and 150,100. Mass Spectrometry. The mass to charge ratio (rn/z)of the electrospray-generated ions of peptides and proteins was determined by a Finnigan MAT TSQ-700 triple-quadrupole mass spectrometer equipped with a 20-keV conversion dynode for enhanced sensitivity. The ion signals were recorded by Finnigan ICIS data system operated on a DECstation 5000/120 that also received signals directly from the UV detector. For LC/MS experiments, only the first of the three quadrupoles was used to performthe m/z analysisand data were recorded in the centroided mode of acquisition. The electrospray needle assembly (Figure 1)consists of three concentric tubes.aa89 The sample solution passed through the innermost of the stainless steel tubes (i.d. 0.004 in., 0.d. 0.008 in.), the sheath liquid through the second concentric tube (i.d. 0.010 in., 0.d. 0.019 in.) and the sheath gas through the outermost (39) Jardine,I.;Hail,M.;Lewis,S.;Zhou, J.;Schwartz,J.;Whitehouee, C. M. Proc. 38th ASMS Conf. Mass Spectrom. Allied Topics, Tucson,AZ, 1990, pp 16-17.
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ANALYTICAL CHEMISTRY. VOL. 65, NO. 23, DECEMBER 1, 1993
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I
1-5% acetic acid and methanol. This observation led us to investigate further to improve the quality of electrospray of aqueous T F A solutions. One of the significant fmdings in our study was the requirement of the sample tube (the innermost tube of the spray assembly) to protrude from the sheath liquid tube by -0.5 mm (Figure 1). In the protruded needle assembly, at a sample solution flow rate of 4 pL/min and a sheath liquid flow rate of 3 pL/min, the voltage on the gold cylindrical electrode required for the optimum spray and signal stability was -3.6 to -3.8 kV compared to 4 . 5 kVor higher for thesample tube kept inside thesheathliquid tube.26.28 This observation is significant, because at higher voltages breakdown of electrospray results in unstable and weak ion signals. With sample tube protruding, the electrospray ionization of analyte solutions containing aqueous 0.1% TFA can be obtained at -3.8 kV on the cylindrical electrode with flow rates as high as 30 pL/min. By contrast, other users of similar sources kept the sample tube 0.5-2 mm inside of the liquid sheath tube to create a “mixing zone”:.25.W so that sample solution and the sheath liquid were mixed before the occurrence of the electrospray. With the exception of Griffin et al.28 these investigators did not examine ESI in aqueous 0.1 % TFA (without the presence of any organic solvent). The nature of the stability of the spray or analyte ion signal when the effluent was 100% aqueousO.l% TFA wasnot discussed in the Griffin’sreport.” The mixing zone was not found to be essential in our investigations, even in the presence of TFA. The observation of better signal stability and higher sensitivity with the protruded sample tube compared to that for the tube kept inside can be explained by the achieved field at the spray tip for a given applied voltage. The outer diameter of the sample tube (0.008 in.) is smaller than that of the sheath liquid tube (0.019 in.). Therefore, the electric field at the tip of the former is higher for a given applied voltage. As reported previously by 0thera,3~J~ at high electric field the electrospray of solutions with high surface tension and conductivity may occur before the onset of the spray destabilizationdischarge. By specially preparing sharp spray tips, Chowdhury and Chait” were able to spray cytochrome c solutions in pure water in ambient air without the assistance from sheath liquid3 or sheath gasf The present finding is in agreement with the earlier report.33 We also observed that the use of a sheath gas further improved the signal stability and sensitivity. The highest gas flowrate that can be obtained from the gas delivery system provided by the manufacturer (Finnigan MAT) is 60 mL/ min. This highest flow rate of nitrogen at a pressure of 20 psi was used in all experiments. To maximize the effect of the sheath gas, the protrusion of the sheath liquid tube from the needle assembly body (Figure 1)was reduced to 0.5 mm from the 1-2 mm that was suggested by the manufacturer (Finnigan operator’s manual, electrospray ionizationsystem, pp 6 8 , 7W1-97036). The reduction in the protrusion was essential to achieve a stable electwspray of TFA solutions at higher flow rates. Thus, the combination of higher field at low applied voltage, reduction in surface tension by adding sheath liquid, and an assistance from the sheath gas were all necessary components to obtain electrospray ionization mass spectra of peptides and proteins in 0.1% TFA solutions at flow rates up to 30 pL/min. The stability of electrospray was examined by monitoring the fluctuation in the total ion current detected by the mam spectrometer. The reconstructed ion chromatograms(RICs) or, preferably, the total ion currents obtained from the electrospray ionizationmass spectraof 5 pmoVaL bradykinin solution infused at the flow rates of 2,10, and 30 aL/min are shown in Figure Za-c, respectively. The mass spectrometer
-
Sample Tuhe
Nerdlo Arromhly BMlY
Shmh Tuhe
Flgure 1. Schematic diegram 01 the ESI ion source. me optimized setup of Um needle assembly f a LCMS using sheath liquid is shown In the bottom panel.
t u b e t h e needle asaemhly body (i.d. 0.0225 in.). 2-Methoxyethanolwas used as sheathliquid,theflow rate of which depended on the flow rate of the effluents entering the ion source. Dry nitrogen generatedfrom liquid nitrogen was used both as sheath gas at a flow rate of 60 mL/min and as drying gas; the temperature and flow rates of the latter varied depending on the flowrate and the composition of liquids received by the source. The flowrates of the gases were read off the flowmeters mounted on the ESI mntml unit. The needle asaembly is operatedat ground potential. The gold-platedcylindricalelectrode around the spray assembly and the transport capillary tube were floated at -3.6 to -3.8 kV, dependingonthe flow ratesof effluentsand theneedletransport capillary distance. A typical distance was 2.2 cm.
RESULTS AND DISCUSSION Optimization of t h e Electrospray Ion Source. The optimization of the source parameters, counter electrode glasscapillarydistance,appliedvoltage onthegoldcylindrical electrode, flow rates of drying and sheath gases, temperature of the drying gas, flow and the nature of the sheath liquid, etc., were performed using infusion electrospray ionization of5pmoVpLbradykininsolutioninO.l%TFAinfusedduectly into the ESI source. We observed that the electrospray of the analyte solutions in aqueous 0.1% TFA at flow rates 1-5 pL/min by direct infusion in the described source cannot be satisfactorily perfomedwithout the use of asheathliquid. Out of the three liquids, methanol, propan-2-01, and 2-methoxyethanol, examined, the latter provided the beat signal stability and sensitivity. Even in the presence of a sheath liquid the signal was not as stable and intense when compared to the one that can be obtained from the infusion of the analyte solution of same concentration, prepared inan equalmixture of aqueous
ANALYTICAL CHEMISTRY, VOL. 65, NO. 23, DECEMBER 1, 1993 3581
- --
RIC
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-
1
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100
200
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Flgun 2. Reconstructed ion chromatogram (RIC) or the total ion cwent obtalnedin the electrogpray Ionbation of 5 pMbradykinin solution
in0.1% TFAat(a)2,(b)10,and(c)30pL/mln.Themassspectrometer was scanned from mlz 310 to 1010 in 2 8. The electrospray process was monitored for 500 s. The sheath liquid flow rates were (a)2, (b) 5.5, and (c) 5.5 pl/mln.
was scanned from m/z 310 to 1010 in 2 8. The flow rates of sheath liquid, 2-methoxyethanol, were 2, 5.5, and 5.5 rL/ min, respectively. Because the flow rates delivered by one of the syringe pumps (Sage Instrumenet) increased in steps, the flow rates of the sheath liquid were not varied continuously to identify the best conditions at higher flow rates. The flow rates of sheath gas in all flow rates were 60 mL/min, the upper limit of the flowmeter. The variation in the RIC at 2, 10, and 30 pL/min were approximately &15%,*13%, and f 2 2 % (Figure 21, respectivley. The average values of RIC were nearly the same at all three flow rates. We believe the variation at higher flow rates could be reduced by using higher flow rates of sheath and drying gases and tuning for better sheath liquid flow rates. We are currently investigating the effects of these parameters on sensitivity and signal stability in ESI LC/MS at higher flow rates. The maas spectraat the three flow rates are shown inFigure 3a-c. To avoid any bias due to fluctuations in intensity at a single scan, scans from 100 to 105 (six scans) were summed for all cases. At flow rates of 2 and 10 pL/min, (M+ 2HI2+ ions of bradykinin (m/z531) were the most intense (panels a and b of Figure 3, respectively). The intensity of m/z 531 decreased from 4.4 X l@to 3.9 X 105 for the increase of flow rate from 2 to 10 pL/min. At 30 pL/min (Figure 3c), the intensity of m/z 531 ion was 8 X 10.'. However, other ions with m/z 323,381, and 399 became intense compared to m/z 531. These additional ions were identified to be the adduct ions of sheath liquid, 2-methoxyethanol (X).Thus, m/z 323, 381,399, and 475 are respectively (4X + H2O + H)+, (5X + HI+, (5X H20 H)+, and (6X+ H2O HI+. It is known that at higher flow rates larger droplets are formed during electrospray. We believe the flow rates and the temperature of the drying gas used in the present experiment were insufficient to assist rapid evaporation of solvents and other liquids from these relatively larger droplets before being analyzed and detected. Unfortunately, we are limited to flow rates of 60mL/min and achievable temperature of 240 OC at these flow rates. The resulta shown in Figures 2 and 3 clearly demonstrate the feasibility of obtaining electrospray ionization mass spectra at flow rates up to 30 pLlmin from aqueous solutions containing 0.1 5% TFA. LC/MS of Standard Peptide Mixtures. The performance of the optimized electrospray ionization source in LC/ MS mode was examined using a solution of seven peptides: d e s - m l - l e u enkephalin, bradykinin, angiotensin 11, bomb-
+
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Flguro 9. Electrapray loniratbn mass spectra of 5 bradyklnin solution In 0.1 % TFA at (a) 2, (b) 10, and (c) 30 pUmkr. The mass spectrometer was scanned from mlz 310 to 1010 In 2 8. Scans from 100 to 105were summed. The (M 2 H P ion represents the doubly protonated lon of bradyklnin. The sheath IIqM flow rates were (a)2, (b) 5.5, and (c) 5.5 pL/min.
+
esin, renin substrate, ACTH (1-241, and B-malenocyte stimulating hormone. The concentration of each in the mixture was 20 pmol/pL with the exception of des-Tyrl-Leu enkephalin (30 pmoVpL) prepared in 0.1% TFA. A 10-pmol aliquot of each (15 pmol of des-Tyrl-Leu enkephalin) was injected to the HPLC column through a 0.5-pL injection loop. The molecular weight of the peptides ranged from 392 for des-Tyrl-Leu enkephalin to 2934.6 for ACTH (1-24). The UV absorption profile and RIC obtained from the LC/ MS analysis of the peptide mixtures are shown in Figure 4. The peaks appeared in the RIC -80 s after they passed through the UV detector at a flow rate of 4 pL/min. No background subtraction or smoothing was done on the RIC shown in Figure 4. With the exception of des-Tyrl-Leu enkephalin (peak 11, the intensities and peak profiles of the peptide ions in the RIC are similar to that of the W trace. The RIC intensity of the ion peak from des-Tyrl-Leu enkephalin is smaller than that of the spectrophotometric absorption peak (upper trace). It was reported earlie+ that low molecular weight peptides (one to three amino acids long) in ESI are formed/detected with less intensities when compared to larger ones if they are present in equal amounta. The present observation supports the earlier reportem The identification of the peptides in each peak, the observed masses, and those calculated from their sequence are given in Table I. Two peptides, angiotensin I1 and ACTH (1-24) (peak 4) eluted without separation under the LC gradient and flow rate (4 pL/min) conditions used. It is importaut to (40)Chowdhwy,S.K.;Kath, V.,and Chait, 8.T.Biochem. Biophys. Res. Commun. 1990,167,686-692.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 23, DECEMBER 1, 1993
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. . , . . , , . . , , . . , , , . . . , . . , . . . . ,
Flgun 4. Ultraviolet absorption intensity of the LC effluentsof a mixture of seven pept#es (upperpanel)and the reconstnrctedIon chromatogram (RIC) following their lonlzatlon by electrospray (bottom panel). The
absclssarepresentsscan nwnbersandelutlontimes. Thepeaksappear In the RIC -80 s after they are being observed In the UV detector. The Mentlflcatlon of the peaks in the two chromatograms is given In Table I. Sheath IIquM (2-methoxyethanol)flowrate was 3 bL/mln (see text).
13:20
1o:oo
Tlml
16:40
. , . . , . , I 2000
Figure 5. (a)Ion chromatogram corresponding to the (M + 2H)2+ lon of bradykinin and (b) that corresponding to the (M + 2H)1+ ion of bombesin; (c)UV-absorptlon profile obtained in the LC/MS of 500 fmol (each) of the two peptides. The mass spectrometer was scanned from mlz 410 to 1710 in 2 8. The chromatographic condltlons are the same as those in Figure 4. lw
1
8,15
Tlyplic digest bwine cytorhmrne C RIC
a)
A.
12113
[ E+O5 4.818
Table 1. Identification of the Peaks in the UV Trace and RIC (Figure 4) and the Observed and Calculated Molecular Masses of the Seven Peptides Examined peak no. 1 2 3 4 4 5 6
peptide des-Tyrl-Leu enkephalin bradykinin @-malenocytestimulating hormone angiotensin11 ACTH(1-24) bombeaii renin substrate (horse)
calcd mol mass (u) monoisotopic average
obsd mol maes (u)
392.2 1059.6 2175.0
392.5 392.0 1060.2 1059.8 2176.4 2175.7 k 0.3
1045.5 2931.6 1618.8 1757.9
1046.2 2933.5 1619.9 1759.1
1045.9 f 0.2 2932.9 0.7 1619.4 0.3 1758.0 0.5
* * *
note that a complete separation of all components is not necessary in the LC/MS measurements provided their molecular masses differ by several mass units. The unresolved Components in the LC can be unambiguously detected in the mass spectra, as was the case for peak 4. The mass spectra obtained by summing scans corresponding to each peak in the RIC yielded very strong peptide ion signals (data not shown). Even a single-scan mass spectrum is sufficientlyintense to provide identificationof the peptides. An examination of the molecular weights measured from the mass spectra (Figure 4) and those calculated from their sequence suggests that the measured molecular masses of small peptides of
