Anal. Chem. 1994,66, 3659-3663
Sensitive and Selective Determination of Proteins with Electrospray Ionization Magnetic Sector Mass Spectrometry and Array Detection Joseph A. Loo' Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48 105 Reinhoid Pesch Finnigan MA T, Barkhausenstrasse 2, Bremen, Germany
A position- and time-resolved ion counting (PATRIC) array detector combined with electrospray ionization and a magnetic sector mass spectrometer was used for sensitive detection of multiply charged protein ions. The array detector's ability to discriminate against lower charged, lower molecular weight ions allows for the collection of a mass spectrum for bovine ubiquitin (8.5 kDa) consuming approximately 90 amol during the data acquisition time. Interfering background ions can be attenuated relative to more highly charged ions by reducing the voltage applied to the dual microchannel plates. This principle is demonstratedwith an electrospray ionization mass spectrum of carbonic anhydrase (29 kDa) in the presence of 0 0 2 % Triton X-100.
are becoming more popular because of their special performance advantages. As the field of electrospray ionization MS has matured, sensitivity limits have improved. Consumption of low femtomole amounts of protein to obtain a mass spectrum has been demonstrated with most types of mass spectrometric a r r a n g e m e n t ~ . ~ ! ~ JInjection ~ J ~ - ~ ~of 500 amol of protein by capillary electrophoresis and ESI analysis with a quadrupole mass spectrometer has been reported. l 5 Recently, impressive low femtomole sensitivity with a double-focusing instrument and array detection has been reported.16 With double-focusing mass spectrometers, focal plane array detectors have improved detection limits for large, singly charged polypeptides. These advantages can also be realized for detection of multiply charged ions. Electrospray ionization (ESI) has advanced the applicabilIn this report, we present preliminary results demonstrating ity of mass spectrometry (MS) to large biomolecule analyses.lq2 the unique characteristics of a position- and time-resolved ion The ability to produce multiply charged molecules has enabled counting (PATRIC) scanning array detector for ESI detection the analysis of large biomolecules of over 150kDa with limited and show several analytical applications that take advantage mass-to-charge ( m / z ) range mass spectrometer^.^ Because of the array detector. Its ability to discriminate against ions of the ease of interfacing such devices, quadrupole mass on the basis of charge allows for detection of proteins to the analyzers are by far the most common ESI d e t e c t o r ~ . ~ - ~ , low ~ attomole level. Detection of higher charged protein However, magnetic s e ~ t o r , time-~f-flight,g.~ ~-~ and ion trapping molecules in the presence of higher concentration, lower instruments (ion trap MSIOJ1and Fourier transform MS12-14) molecular weight contaminants will also be demonstrated. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S . F.; Whitehouse, C. M.Science 1989, 246, 64-71. (2) Smith,R.D.;Loo, J.A.;Edmonds,C. G.;Barinaga,C. J.;Udseth,H. R.Anu1. Chem, 1990,62, 882-899. (3) Feng, R.; Konishi, Y. Anal. Chem. 1992, 64,2090-2095. (4) Smith, R. D.; Loo, J. A.; Ogorzalek Loo, R. R.; Busman, M.; Udseth, H. R. Muss Spectrom. Rev. 1991, 10, 359451. ( 5 ) Larsen, B. S.; McEwen, C. N. J.Am. SOC.Muss Spectrom. 1991,2,205-211. (6)Cody,R. B.;Tamura,J.;Musselman,B. D. Anal. Chem. 1992,64,1561-1570. (7) Loo, J. A.; Giordani, A. G.; Muenster, H. Rapid Commun. Muss Spectrom. 1993, 7, 186-189. (8) Verentchikov, A. N.; Ens, W.; Standing, K. G. Anal. Chem. 1994.66, 126133. (9) Mirgorodskaya, 0.A,; Shevchenko, A. A,; Chernushevich, I. V.; Dodonov, A. F.; Miroshnikov, A. I. Anal. Chem. 1994, 66, 99-107. (10) Van Berkel, G. J.; Glish, G. L.; McLuckey, S . A. Anal. Chem. 1990, 62, 1284-1295. (11) Schwartz, J. C.; Syka, J. E. P.; Jardine, I. J. Am. SOC.MussSpecrrom. 1991, 2 , 138-204. (12) Henry, K. D.; McLafferty, F. W. Org. Muss Spectrom. 1990, 25, 490492. (13) Winger, B. E.; Hofstadler, S . A.; Bruce, J. E.;Udseth, H. R.; Smith, R. D. J . Am. SOC.Muss Spectrom. 1993, 4, 566-577. (14) Guan, Z.; Hofstadler, S . A,; Laude, D. A., Jr. Anal. Chem. 1993.65, 15881593.
0003-2700/94/0366-3659$04.50/0 @ 1994 American Chemlcal Society
EXPER I MENTAL SECT1ON ESI-MS analyses were performed with a Finnigan MAT 9004 forward geometry hybrid (EBqQ) mass spectrometer (Bremen, Germany) equipped with a conventional 20 kV conversion dynode/secondary electron multiplier (SEM) point detector and a PATRIC focal plane detector after the magnet and before the tandem quadrupole section? A pair of deflector plates after the magnet is used to deflect the ion beam to the array detector mounted underneath the SEM detector. Switching between the two detectors is done electronically and can be performed in less than 0.5 s. For operation with the PATRIC detector, an 8% m / z range of the m / z centered (15) Wahl, J. H.; Goodlett, D. R.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1992, 64, 3194-3196. (16) Cody, R. B.; Tamura, J.; Finch, J. W.; Musselman, B. D. J. Am. SOC.Muss Spectrom. 1994, 5 , 194-200.
Analytical Chemistry, Voi. 66, No. 21, November 1, 1994 3650
on the detector was used. Mass resolution with the array detector can be generally greater than 5000 (full-width at half height) and up to approximately 9000. Thevoltage across the front and back of the microchannel plates is designated as V ~ c p . Analyses were performed at full accelerating potential (5 kV). Mass spectra were acquired at 10s decade-’. An electrospray ionization interface based on a heated glass capillary (0.5 mm i.d. X 6.2 mm 0.d. X 218 mm length) inlet was used.’ Droplet desolvation was accomplished by a countercurrent stream of warm nitrogen (-60 “C) and by collisions in the electrospray interface controlled by adjustment of thevoltagedifference between the tube lens at themetalized exit of the glass capillary and the first skimmer element. A gentle stream of SF6 coaxial to the spray suppressed possible corona discharges. All biological samples were obtained from Sigma Chemical Co. (St. Louis, MO) and were used without further purification. Samples were prepared in the appropriate solvent and buffer systems and infused through the ESI source at a flow rate of 0.5-1.5 pL min-l. The fused silica and metal capillaries used for sample introduction were rinsed with acetic acid and water/methanol/2.5% acetic acid solutions to remove any adsorbed materials prior to analysis of the low protein concentration solutions. ESI-MS analysis of a “blank” sample was also performed to confirm that all adsorbed protein material was adequately removed.
RESULTS AND DISCUSSION The principles and operation of the PATRIC array detector for determining the m / z of an ion and for scanning operation have been previously discussed17J8and are beyond the scope of this report. However, the unusual ability of the PATRIC array detector to discriminate against ions on the basis of charge state has not been reported, nor have its analytical applications. This discrimination is based on the different yield of secondary electrons at the surface of the detector, a microchannel plate (MCP), for ions of different charge state. Friedman and c o - w ~ r k e r s ~have ~ ~ * measured ~ secondary electron yields as a function of ion velocity and mass. The PATRIC detector is an ion counting detector whose position determination is based on the direct evaluation of the number of secondary electrons collected on a special collector plate. These secondary electrons are generated by ions impinging onto the surface of the first of two microchannel plates in chevron arrangement. The typical gain of the two MCPs is lo6 at a VMCPof about 900 V per MCP, yielding a cloud of lo6 electrons for each ion detected. The read-out mechanism is designed to handle electron clouds of about 104-106 electrons per event. This average number of electrons can easily be adjusted by changing VMCP.In order to function properly, the position sensing electronics applies a bandpass filter to the number of secondary electrons prior to evaluation. If the number of electrons is above or below this level, the (1 7) Evans, S.In Mefhods in Enzymology: MassSpecfromefry;McCloskey, J. A., Ed.; Academic Press: San Diego, CA, 1990; Vol. 193, pp 61-86. (18) Pesch, R.; Jung, G.; Rost, K.; Tietje, K.-H. Proceedings of fhe 37fh ASMS Conference on Mass Specfrometry and Allied Topics, Miami Beach, FL, 1989, American Society for Mass Spectrometry: East Lansing, MI, 1989; pp 1079-1080. (19) Beuhler, R. J.; Friedman, L. Nucl. Instrum. Methods 1980, 170, 309-315. (20) Xu, Y.; Bae, Y. K.; Beuhler, R. J.; Friedman, L. J . Phys. Chem. 1993, 97, 11883-11886.
3660
Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
determination of pulse heights would be less reliable, either by saturating the A/D conversion (too many electrons) or by having not enough precision (too few electrons). Only events whose numbers of secondary electrons fall between a given minimum and maximum number are evaluated. For singly charged ions and ions having only a few charges, the secondary electron yield of the microchannel plates does not vary enough to show any noticeable difference between ions. VMCPneeds to be set to about 850-950 V in order to have the overall number of secondary electrons fall within the bandpass. Highly charged ions, however, generate many more secondary electrons upon hitting the microchannel plate than do, for example, singly charged ions. This is due to the very high kinetic energy of these ions. With an acceleration voltage (Vas) of 5 kV and a postacceleration of 2 kV at the detector (ZVMCp, where VMCP is 1000 V), a singly charged ion hits the detector surface with 7 keV of kinetic energy, whereas an ion carrying 50+ charges impinges the surface with 350 keV. The latter ion releases significantly more secondary electrons than the former. Such highly charged ions release so many secondary electrons upon impact that more than 106 electrons per ion strike the collector plate if VMCPis at its normal operating value. Hence, the PATRIC electrons will not register these events as valid events. Consequently, there is a discrimination of highly charged ions when VMCPis set for optimum detection of singly charged ions, and vice versa. Therefore, in order to detect highly charged ions, VMCPmust be reduced to decrease the number of secondary electrons to a level between the minimum and maximum threshold. The optimum VMCPcan be as low as 600 V. This behavior leads to different optimum operating conditions for detection of singly and highly charged ions. For example, with a 3.0 fmol pL-’ solution of bovine ubiquitin (MI 8565), the ESI mass spectrum with VMCPat +825 V shows poor sensitivity for the multiply charged protein analyte (Figure la). Lowering V ~ c to p +690 V for a 0.3 fmol pL-l solution yields the spectrum in Figure l b (the sum of three 6-s scans). Approximately 90 amol was consumed during the time spent recording the lower mass spectrum. Figure 2 shows the electrospray mass spectrum for bovine serum albumin (66 kDa), consuming 47 fmol during the data acquisition time. Full scan spectra ( m l z 500-3000) can be collected from less than 5 fmol consumed of bovine albumin and less than 10 fmol of albumin dimer (133 kDa) with a signal-to-noise ratio greater than 7:l for the largest relative abundance peak. Similar performance can be obtained from a 2 fmol pL-I solution of porcine pepsin (negative ion ESI, 34 kDa), consuming less than 500 amol. Although low attomole “sensitivity limits” are demonstrated, this sensitivity may not accurately reflect the actual limit of the entire methodology, as described by Cody and co-workers.16 For example, the consumption of 90 amol in Figure l b does not take into account the amount used to tune the ion source prior to data acquisition, etc. However, for the purposes of this communication, to describe the analytical advantages of the PATRIC array detector, a low attomole detection limit is reported. No special protocols were used to handle such dilute protein solutions. The low detection limit is enhanced by the array detector’s ability to discriminate against low molecular weight
100,
80-
ml z Figure 1. Eiectrospray ionization mass spectra of bovine ubiquitin (M, 8565) at (a) a concentration of 3.0 fmol pL-I and Vwp of +825 V (consuming 2.7 fmol during the total acquisition time) and (b) a concentratlon of 0.3 fmol pL-I and V , of +690 V (0.09 fmoi consumed). The solvent system used was 2:l MeOH:H20with acetic acid added to the 2.5% (vlv) level. The solution flow rate was 1 pL min-I. l”0,
45+
1 148
I
mlz Flgure 3. €SI mass spectra of a six-component mixture at a concentrationof 1 p m i pL-I for each component (1pL mln-l), consisting of substance P (0,M, 1348),bovine insulin (A,4 5734),horse heart cytochrome c (M, 12 360),hen egg white lysozyme (*,M, 14 306), bovine carbonicanhydrase (CA, M, 29 021),and bovine serum albumin (BSA, M, 66 430),with V , at (a) +850 and (b) +625 V.
’“9
have dramatic effects in the relative abundances of ions. The (M + 5H)5+ molecule of insulin is approximately half the relative abundance of the (M 1 lH)ll+ lysozyme ion with VMCPat +675 V. Increasing VMCPto +700 V increases the [insulin5+]/[lysozyme1*+]ratio to 1.0; the ratio increases to 1.4 at a VMCPof +750 V. For bovine albumin, the highest charged ion detected with V ~ c at p +625 V is 65+; the 62+ charge state is resolved at +700 V, and the 54+ charge state is the highest charged ion detected at +925 V. In a given m / z region for a mixture sample, the higher charged ion is selectively detected at lower MCP voltage. It is difficult to determine the effects of only mass (as opposed to mass-to-charge ratio) at a constant kinetic energy (or charge). In general, the larger the peptide or protein, the greater the average charge state. In Figure 3, the optimum VMCPvalues for two similarly sized proteins, cytochrome c (12.3 kDa) and lysozyme (14.3 kDa), aredrastically different. Because cytochrome c has an apparent higher average charge state in an acidic pH solution, its optimum V ~ c isp lower than that for lysozyme. Is it also difficult to determine the dependence of detector selectivity on ion kinetic energy. It is known that the secondary electron yield of ions hitting a surface depends on the type, mass, and velocity of the ions.19!20 In changing V ~ c p one , also varies the ion kinetic energy as well as the electron gain of the MCPs. However, it is still interesting to report that the 45+ charge state ion of carbonic anhydrase (29 kDa) disappears upon increasing VMCP from +615 to +650 V, which corresponds to a kinetic energy difference of approximately 3.2 keV and an increase in the microchannel plate’s secondary electron gain of about 2.*l Approximately the same kinetic energy difference was found for cytochrome c (e.g., the 19+
+
mlz Flgure 2. ESI mass spectrum of bovine serum albumin (66 kDa) with Vwp of +650 V. Approximately 47 fmoi of protein was consumed during the period needed to acquire the data.
background contaminants. By using lower VMCPvoltages, highly charged protein molecules are preferentially detected. This ability to discriminate against lower charged species is further demonstrated by the spectra in Figure 3. An equimolar mixture of six polypeptide components, ranging in molecular mass from 1348 (substance P) to 66 430 Da (bovine serum albumin) was analyzed by ESI-MS with the PATRIC detector. With V ~ c pat +850 V (Figure 3a), ions for substance P(2+ charge state), bovine insulin (3+ to 6+), and hen egg white lysozyme (9+ to 12+) were preferentially detected (although cytochrome c ions are also evident in low relative abundance). Lowering VMCPto +625 V allows for “selective” detection of the higher charged molecules from the three remaining components (Figure 3b). Discrimination based upon charge state is easily observed from the example in Figure 3. Small changes in VMCPcan
Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
3681
741
785
40:
23-
600
Boa
1330
12b0
14c0
mlz Figure 4. Eiectrospray ionization mass spectra of 3.4 pmoi pL-I of bovine carbonic anhydrase with 0.02% Triton X-100 (reduced) in 2:l MeOH:H20and 2.5% acetic acid wlth ,V at (a) +750 and (b) +635 V. Approximately 7.9 pmoi of protein was consumedduringacquisition of spectrum b.
charge state ion is “lost” by increasing VMCPfrom +650 to +725 V, or a 2.8 keV kinetic energy difference, along with a factor of 5 increase in MCP gain). The presence of salts and buffers can often be disabling for electrospray ionization MS experiments. However, the advantages of low molecular weight (low charge) discrimination can be realized for high levels of interfering buffers and other additives used in protein chemistry. For example, Triton X- 100 (reduced), a nonionic polyoxyethylene detergent, is often used to solubilize proteins. ESI mass spectra for a 3.4 pmol pL-1 solution of bovine carbonic anhydrase (29 kDa) in the presence of 0.02% (w/v) Triton X- 100 are shown on Figure 4. As expected, the mass spectrum with VMCPat +750 V (Figure 4a) shows only singly charged ions for the Triton X-100 oligomers, whereas reducing VMCPto +635 V allows detection of the carbonic anhydrase multiply charged molecules. Broadening of the peaks is evident, most likely from Triton X-100 adduction to the protein.22 ESI mass spectra of proteins in the presence of 50 mM Tris [tris(hydroxymethy1)aminomethanel have also been obtained. Ion discrimination has been previously reported with other types of instrumentation. Hofstadler et al.23 varied the bandpass kinetic energy filter for selecting ESI-produced ions for Fourier transform MS detection by adjusting the ion trapping potentials. The selection of trapped ions was predominantly based on the m / z of the ions. Feng and K o n i ~ h filtered i ~ ~ ions entering the mass analyzer of a triple quadrupole instrument based on kinetic energy. This method (21) Wiza, J. L. Nucl. Insfrum. Merhods 1979,162, 587-601. (22) Ogorzalek Loo, R. R.; Dales, N.; Andrews, P. C. Proceedings of the 41sf ASMS Conference on Mass Specrrometry and Allied Topics, San Francisco, CA, 1993; American Society for Mass Spectrometry: Santa Fe, N M , 1993; p 581. (23) Hofstadler, S. A.; Beu, S. C.; Laude, D. A. Anal. Chem. 1993,65,312-316.
3882 Analytical Chemistry, Vol. 66,No. 21, November 1, 1994
was later used by Covey and Douglas25 to measure collision cross sections of highly charged ions. The results presented in this communication are a consequence of an upper pulse height cutoff set too low and a too-small dynamic range for pulse counting applications with electrospray ionization, especially for large, multiply charged ions. It would be difficult to find experimental parameters that would minimize the charge discrimination effects of the PATRIC detector for multiple component mixture analysis. Individual scans acquired with different VMCpvalues could be collected to provide, qualitative information for a mixture sample, as demonstrated in Figure 3, but quantitative analysis would be problematic. Alternatively, it is possible to step VMCPautomatically under computer control through the applicable range of voltages to obtain the optimum conditions for all ions. Under these conditions, the quantitative results of the ion counting can be expected to be even more reliable than those with analog registration. The effect of different secondary electron yields applies to all SEM-type detectors,19,20J6-27 resulting in significantly different responses for different multiply charged ions. For the present time, the large dynamic range of the conventional SEM detector of the mass spectrometer system is used for complex mixtures, and the PATRIC array detector is used for high sensitivity analyses and for applications where charge discrimination is beneficial. The observations presented should be general for pulsecounting detection in ESI-MS and should not be limited to the type of mass spectrometer (Le., magnetic sector instrument). Quadrupole mass spectrometers based on ion counting detection can be set up to preferentially detect higher charged ions by adjusting the low pulse height threshold leve1.28 Sundqvist and co-workers have measured the seondary electron yield of electrospray-generated ions impacting a detector and have also applied the discriminating nature of their detector (for a method they term “secondary electron resolved mass spectrometry” or “SERMS”).27 It can be expected that the discrimination phenomenon can be observed with all detectors that are capable of applying either a threshold or a bandpass prior to the further processing of secondary electron pulses generated by ions hitting the detector surface. These detection limitations can be turned into advantages for the right applications.
CONCLUSION This preliminary report represents the first example of detector discrimination of multiply charged ions produced by ESI with a magnetic sector instrument and presents some useful analytical applications of the technique. Sensitivity limits of large, multiply charged ions with an array detector that has a maximum pulse height limit can be dramatically improved by lowering the electron gain of the microchannel plate. Semiquantitative mixture analysis is difficult because each analyte may have different optimum array voltages. (24) Feng, R.; Konishi, Y. Proceedings of the 39rh ASMS Conference on Mass Specrrometry and Allied Topics, Nashville, TN,1991; American Society for Mass Spectrometry: East Lansing, MI, 1991; pp 260-261. (25) Covey, T.; Douglas, D. J. J . Am. Soc. Mass Spectrom. 1993, 4, 616-623. (26) Alexandrov, M. L.; Gall, L. N.; Krasnov, N . V.; Lokshin, L. R.; Chuprikov, A. V. Rapid Commun. Mass Spectrom. 1990, 4, 9-12. (27) Axelsson, J.; Reimann, C. T.; Sundqvist, B. U. R. Inr. J . Moss Specrrom. Ion Processes 1994, 133, 141-155. (28) Light-Wahl, K. J.; Loo, J . A.; Smith, R. D., unpublished results.
However, the ability to discriminate against lower charged species allows one to “tune” a spectrum for a higher charged, low level species in a complex mixture.
ACKNOWLEDGMENT We wish to thank Jeffrey W. Finch (JEOL USA) for an early copy of his group’s manuscript (ref 16), Igor Chernuschevich (University of Manitoba) for discussions describing his group’s results30and for bringing to our attention the work (29) Bondarenko, P. V.; Grant, P. G.; Macfarlane, R. D. Presented at the 42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29-June 3, 1994; paper WP 212. (30) Standing, K. G.; Chernushevich, I.; Ens, W.;Verentchikov, A. Presented at the 42ndASMS Conferenceon MassSpectrometryand Allied Topics, Chicago, IL, May 29-June 3, 1994; paper FOA 9:30.
of the Uppsala group (ref 27), and Rachel R. Ogorzalek Loo (University of Michigan) for the detergent sample and for many helpful discussions. Note Added in Proof. After the original submission of this manuscript, work from two other laboratories was presented at the recent ASMS Conference on Mass Spectrometry and Allied Topics (May 29-June 3, 1994, Chicago, IL) that described charge discrimination effects with pulse counting detection and time-of-flight mass spectrometry and their applications with 252Cfplasma desorption29and electrospray ionization .30 Recelved for review April 12, 1994. Accepted July 15, 1994.” @Abstractpublished in Aduance ACS Abstracts, September 1, 1994.
Analytical Chemistry, Vol. 66,No. 21, November 1, 1994
3663
