Analysis of a Tryptic Digest of Pig Hemoglobin Using ESI-FAIMS-MS

tides of pig hemoglobin were examined by ESI-FAIMS-. MS using a newly designed FAIMS device. The new, hemispherical geometry of the inner electrode ...
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Anal. Chem. 2000, 72, 4577-4584

Analysis of a Tryptic Digest of Pig Hemoglobin Using ESI-FAIMS-MS Roger Guevremont* and David A. Barnett

Institute for National Measurement Standards, National Research Council of Canada, Ottawa, Canada K1A 0R6 Randy W. Purves and John Vandermey

PE Sciex, Concord, Canada L4K 4V8

The continuous gas-phase ion separation and atmospheric pressure focusing properties of high-field asymmetric waveform ion mobility spectrometry (FAIMS) offer significant advantages for the mass spectrometric analysis of tryptic digests of proteins. In this study, tryptic peptides of pig hemoglobin were examined by ESI-FAIMSMS using a newly designed FAIMS device. The new, hemispherical geometry of the inner electrode served to deliver the ions, via the gas flows, to the center axis of the FAIMS analyzer, improving the sensitivity relative to previous prototypes. Mass spectra collected using this new FAIMS showed significantly less chemical background noise than conventional ESI-MS, while maintaining approximately the same absolute sensitivity as that observed with ESI-MS. As a consequence of the ion separation in FAIMS, the identification of the tryptic fragments was simplified and some peptides, such as the triply protonated VVAGVANALAHK3+, that were obscured by the intense background of ESI-MS, were readily detected using ESI-FAIMS-MS. In addition, the FAIMS device was shown to separate isobaric ions at m/z 532.4. Correlations between CV and mass-to-charge ratio, as well as CV and ionic collision cross section, were evaluated for 38 peptide ions identified in the tryptic digest. The correlation between the CV of the peptide and the massto-charge ratio is very poor, indicating good orthogonality between the separation by FAIMS and the separation by mass spectrometry. A little more than a decade ago, mass spectrometric analysis of large biomolecules was revolutionized by the development of electrospray ionization (ESI)1 and matrix-assisted laser desorption ionization (MALDI).2 ESI-mass spectrometry (ESI-MS) has been used in several different areas of protein research, including the investigation of conformational changes,3 the probing of noncovalent interactions,4-6 and in obtaining sequencing information.7,8 The analysis of tryptic digests using ESI-MS to obtain sequence * Corresponding author. e-mail: [email protected]. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299. (3) Hoaglund-Hyzer, C. S.; Counterman, A. E.; Clemmer, D. E. Chem. Rev. 1999, 99, 3037-79. (4) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1-23. 10.1021/ac0000271 CCC: $19.00 Published on Web 08/30/2000

© 2000 American Chemical Society

information has become increasingly important, not only for protein identification but also for the detection of sequence modifications.9 In most instances, biological samples are very complex and the analyte of interest may be present in only small amounts. Consequently, there is an increasing need to “simplify” ESI-mass spectra and lower the limits of detection. Separation methods, such as liquid chromatography (LC) and capillary electrophoresis (CE), are commonly used to simplify spectra. However, chromatographic techniques are time-consuming and can only produce sample ions in short, finite pulses. Furthermore, these methods are still compromised by the intense chemical noise inherent to ESI since the separation occurs prior to ionization. Alternatively, ions can be separated in the gas phase using a technique known as high-field asymmetric waveform ion mobility spectrometry (FAIMS).10-13 FAIMS separates ions at atmospheric pressure and room temperature on the basis of differences in ion mobilities at high vs low electric fields.10,14 Experimentally, this difference in mobility (Kh/K) is reflected in the value of the compensation voltage (CV) at which an ion is transmitted through the device. A detailed description of the separation mechanism in FAIMS has been given elsewhere.10,11,13 The sensitivity of a FAIMS device with cylindrical geometry15 can be high due to an ion-focusing mechanism described by Guevremont and Purves.12 A cylindrical-geometry FAIMS device was characterized using mass spectrometry11 and has been used as an interface between ESI and MS (ESI-FAIMS-MS).13 The application of ESI-FAIMSMS to biochemical samples, such as amino acids,16 peptides,17 and proteins,18 has been investigated. (5) Przybylski, M.; Glocker, M. O. Angew. Chem., Int. Ed. Engl. 1996, 35, 80626. (6) Veenstra, T. Biophys. Chem. 1999, 79, 63-79. (7) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Science 1990, 248, 201-4. (8) Yates, J. R., III J. Mass Spectrom. 1998, 33, 1-19. (9) Humphery-Smith, I.; Cordwell, S. J.; Blackstock, W. P. Electrophoresis 1997, 18, 1217-42. (10) Buryakov, I. A.; Krylov, E. V.; Nazarov, E. G.; Rasulev, U. K. Int. J. Mass Spectrom. Ion Processes 1993, 128, 143-8. (11) Purves, R. W.; Guevremont, R.; Day, S.; Pipich, C. W.; Matyjaszczyk, M. S. Rev. Sci. Instrum. 1998, 69, 4094-105. (12) Guevremont, R.; Purves, R. W. Rev. Sci. Instrum. 1999, 70, 1370-83. (13) Purves, R. W.; Guevremont, R. Anal. Chem. 1999, 71, 2346-57. (14) Mason, E. A.; McDaniel, E. W. Transport properties of ions in gases; John Wiley & Sons: New York, 1988. (15) Carnahan, B. L.; Tarassov, A. S. U.S. Patent 5,420,424, 1995.

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Figure 1. Schematic of the ESI-FAIMS system coupled to a PE Sciex API 300 triple quadrupole mass spectrometer.

FAIMS offers several advantages for the analysis of tryptic digests. First, the separation in FAIMS occurs after the ESI source. Consequently, many of the ions that contribute to the chemical noise inherent to ESI are filtered out by the FAIMS device. Second, the separation in FAIMS is a function of a voltage (CV), whereas techniques such as LC and CE separate compounds as a function of time. The CV can therefore be set for continuous ion transmission to allow for a detailed MS or MS/MS investigation of a particular tryptic peptide. Third, separation of the ions is independent of m/z so that, in some instances, isobaric ions may be distinguished.16-18 Finally, the atmospheric pressure ionfocusing mechanism observed in FAIMS12 means that the dramatic reductions in the chemical noise may be achieved without sacrifice of absolute sensitivity. This study illustrates these advantages for the analysis of peptide fragments resulting from a tryptic digest of pig hemoglobin. EXPERIMENTAL SECTION ESI-MS and ESI-FAIMS-MS. A schematic of the ESI-FAIMSMS instrument is shown in Figure 1. The FAIMS hardware is similar to a previous version used to investigate atmospheric pressure ion trapping using ESI-FAIMS-TOFMS.19 This FAIMS device consists of two concentric cylinders. The end of the inner cylinder (10-mm o.d.) facing the mass spectrometer was machined to a hemispherical surface. The inner surface of the outer cylinder (14-mm i.d.) nearest the mass spectrometer was machined to a concave spherical shape so that the analyzer region was kept constant at a width of 2 mm. The outer surface of the end of the outer cylinder was machined flat with a 1-mm aperture in the center. The outer cylinder made electrical contact with the orifice plate (25 V) of the PE Sciex API 300 triple quadrupole mass spectrometer. The entire FAIMS device was held by a PEEK (16) Barnett, D. A.; Ells, B.; Purves, R. W.; Guevremont, R. J. Am. Soc. Mass Spectrom. 1999, 10, 1279-84. (17) Guevremont, R.; Purves, R. W. J. Am. Soc. Mass Spectrom. 1999, 10, 492501. (18) Purves, R. W.; Guevremont, R.; Barnett, D. Int. J. Mass Spectrom. 2000, 197, 163-77. (19) Guevremont, R.; Purves, R. W.; Barnett, D. A.; Ding, L. Int. J. Mass Spectrom. 1999, 193, 45-56.

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insulating sleeve (not shown) that was attached to the orifice plate by two screws. This geometry of FAIMS has several advantages over an earlier prototype in which the electrospray ionization source was located inside of the FAIMS device and the ions were extracted from an opening located on the side of the outer cylinder of the device.11,13,17 First, this new design enables the ESI source to be placed external to the FAIMS device, thereby allowing high flow rates that are typical in ionspray to be used without danger of contamination of the gas in the FAIMS analyzer region. The desolvation region in the present design, resulting from a countercurrent of gas flowing out through the curtain plate, serves to isolate FAIMS from the neutrals and droplets generated at the electrospray needle. Second, the ions are efficiently sampled by the mass spectrometer since all stable ions in the annular analyzer space are brought to the center axis of the inner electrode and are focused toward the orifice plate of the mass spectrometer. This approach for increasing the sensitivity of FAIMS has been described previously.19 Finally, unlike the previous FAIMS device that was used with an API 300 mass spectrometer,11,13,17 this new FAIMS prototype did not require a custom MS interface. Consequently, switching from ESI-FAIMS-MS to ESI-MS was greatly facilitated. The ESI needle13,17 (4000 V, 80 nA) was positioned at an angle of ∼45 degrees and approximately 1 cm from a 3-mm opening in the curtain plate. The curtain plate was electrically insulated from the outer cylinder of the FAIMS device and biased to 450 V. Nitrogen gas was passed through a charcoal/molecular sieve filter and introduced into the gap (∼1.5 mm) between the curtain plate and the outer FAIMS cylinder at a flow rate of 2 L/min. The gas split into two flows. The larger portion of this gas flowed out of the opening in the curtain plate countercurrent to the arriving electrospray ions, thereby facilitating desolvation. The smaller portion of this gas flow carried the ions inward through a 1-mm opening in the outer FAIMS cylinder and along the analyzer region of the FAIMS device. Under the appropriate electrical conditions of DV and CV, ions were transmitted through FAIMS and focused to a region in front of the spherical tip of the inner cylinder where

they were sampled by the mass spectrometer. There was no net flow through the carrier gas inlet in this study. A MKS Baratron model 170M-6B pressure meter with a pressure sensing head of type 3TOBJ-1000 (MKS Instruments Inc., Boulder, CO) was connected to this port. The pressure in the FAIMS device was ∼0.5-1 Torr below atmospheric pressure. The asymmetric waveform used with FAIMS has been described previously,13,20 however in this study, the relative amplitude of the sinusoidal wave to its harmonic was approximately 4:1. The frequency of the waveform was 750 kHz, and unless otherwise stated, the dispersion voltage (DV) was -3900 V. To detect the tryptic fragments, the CV was scanned from 0 to -16 V in steps of 0.2 V and a mass spectrum was collected at each voltage. Unless otherwise indicated, mass spectra were acquired from m/z 250 to 1100 in increments of 0.1 at a dwell time of 5 ms. Since each spectrum required about 1 min to acquire, the collection of data from CV -16 to 0 (81 mass spectra) required about 90 min. The same acquisition with a TOF mass spectrometer is projected to take less than 3 min, limited only by the practical scan rate of the FAIMS apparatus. Comparisons of ESI-MS and ESI-FAIMS-MS experiments were facilitated by the relative ease of conversion between these modes of operation. ESI mass spectra were collected by removing the FAIMS device from the orifice plate and mounting the original API 300 curtain plate on the instrument. The electrospray source was then operated in the conventional manner. The conversion typically required less than 15 min and did not require breaking vacuum. The ESI and MS operating conditions were identical for both setups. Comparisons of conventional ESI-MS spectra and ESIFAIMS-MS spectra were made using identical single mass scans (i.e., 5-ms dwell time and a step size of 0.1 m/z). A nebulizer gas was not used for any of the ESI-MS or ESI-FAIMS-MS experiments. Preparation of the Tryptic Digest. Tryptic peptides of pig hemoglobin were supplied as a dry powder by Professor David Clemmer, Indiana University (Bloomington). Solutions were prepared by dissolving 7.5 µg/mL of the powder in a solution consisting of 49% water, 50% methanol, and 1% acetic acid. This sample contained the equivalent of about 250 nM in each of the alpha and beta chains of the protein. RESULTS AND DISCUSSION Comparison of Mass Spectra Collected with ESI-MS and ESI-FAIMS-MS. A tryptic digest of pig hemoglobin was analyzed using both ESI-MS and ESI-FAIMS-MS. Figures 2-6 show comparisons of mass spectra obtained using both techniques. With ESI-FAIMS-MS, the particular ions that are observed in a mass spectrum, and their relative abundances, are a function of the applied compensation voltage, CV. For example, Figure 2 shows a comparison of mass spectra collected by ESI-MS (Figure 2a) with those collected by ESI-FAIMS-MS (Figure 2b) for m/z 300400. With FAIMS, the CV (-7.0 V) was selected for optimal transmission of the tryptic fragment HLDNLK2+ at m/z 370.3 (note that for clarity throughout this document, all ions are of the form [M + nH]n+, but are given as Mn+). Two other tryptic fragments, VADALTK2+ at m/z 359.3 and VVAGVANALAHK3+ (20) Viehland, L. A.; Guevremont, R.; Purves, R. W.; Barnett, D. A. Int. J. Mass Spectrom. 2000, 197, 123-30.

Figure 2. Mass spectra of a 7.5 µg/mL tryptic digest of pig hemoglobin collected using (a) conventional ESI-MS and (b) ESIFAIMS-MS with FAIMS operating at a DV of -3900 V and a CV of -7.0 V. Both spectra are 1 scan, 5-ms dwell time, and 0.1 m/z step size.

at m/z 384.1, were also transmitted at this CV, although their transmissions were not optimal. The ion at m/z 363 could not be identified unambiguously. Of the tryptic fragments that are readily apparent in the ESI-FAIMS-MS spectrum, only the m/z 370.3 ion is readily observed in the ESI-MS spectrum. The intensities of the HLDNLK2+ ion in the ESI-MS and ESI-FAIMS-MS spectra are comparable, however as Figure 2 indicates, the signal-tobackground ratio (S/B) for HLDNLK2+ has improved from approximately 10:1 (ESI-MS) to more than 50:1 (ESI-FAIMS-MS). Figure 3 shows a comparison of mass spectra collected by ESI-MS (Figure 3a) and ESI-FAIMS-MS (Figure 3b) for m/z 450-550. In Figure 3b, the FAIMS was set to CV ) -7.8 V, conditions that were optimal for transmission of the tryptic fragment EAVLGLWGK2+ at m/z 487.0. These conditions are also suitable for transmission of MFLGFPTTK2+ at m/z 521.4 (optimal at CV ) -7.2 V), but unsuitable for transmission of TYFPHFNLSHGSDQVK4+ at m/z 470.2. This latter ion is transmitted through FAIMS at very high CV (-13.2 V) and is therefore absent from the mass spectrum collected at CV ) -7.8 V. In Figure 4 (m/z range 550-650), the FAIMS conditions (CV -10.2 V) were selected to transmit TYFPHFNLSHGSDQVK3+ at m/z 626.4. In Figure 5 (m/z range 670-770), the FAIMS was tuned to transmit VGGQAGAHGAEALER2+ at m/z 712.0 (CV ) -6.8 V). Finally, the CV was adjusted in Figure 6 (m/z range 950-1050) for the optimum transmission of FFESFGDLSNADAVMGNPK2+ at m/z 1023.7 (CV ) -6.6 V). In each of Figures 2-6, a comparison of ESI-MS and ESI-FAIMS-MS mass spectra illustrates that the ion separation provided by FAIMS reduced the number of types of ions in the mass spectrum and also resulted in significant reductions in the chemical background noise characteristic of ESI. This has been achieved with little or no loss in absolute sensitivity. The improved S/B is expected to translate into improved detection limits for many of the tryptic peptides. Separation of Tryptic Digest Ions with FAIMS. The optimum transmission of an ion through FAIMS is determined using Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

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Figure 3. Mass spectra of a 7.5 µg/mL tryptic digest of pig hemoglobin collected using (a) conventional ESI-MS and (b) ESIFAIMS-MS with FAIMS operating at a DV of -3900 V and a CV of -7.8 V. Both spectra are 1 scan, 5-ms dwell time, and 0.1 m/z step size.

Figure 5. Mass spectra of a 7.5 µg/mL tryptic digest of pig hemoglobin collected using (a) conventional ESI-MS and (b) ESIFAIMS-MS with FAIMS operating at a DV of -3900 V and a CV of -6.8 V. Both spectra are 1 scan, 5-ms dwell time, and 0.1 m/z step size.

Figure 4. Mass spectra of a 7.5 µg/mL tryptic digest of pig hemoglobin collected using (a) conventional ESI-MS and (b) ESIFAIMS-MS with FAIMS operating at a DV of -3900 V and a CV of -10.2 V. Both spectra are 1 scan, 5-ms dwell time, and 0.1 m/z step size.

Figure 6. Mass spectra of a 7.5 µg/mL tryptic digest of pig hemoglobin collected using (a) conventional ESI-MS and (b) ESIFAIMS-MS with FAIMS operating at a DV of -3900 V and a CV of -6.6 V. Both spectra are 1 scan, 5-ms dwell time, and 0.1 m/z step size.

a compensation voltage scan combined with single ion monitoring MS. This experiment is called an ion-selected CV scan (IS-CV scan). Figure 7 shows IS-CV scans for the m/z 626.4 and 638.0 ions over the CV range from 0 to -16 V, for a 7.5 µg/mL solution of a tryptic digest of pig hemoglobin. The m/z 626.4 ion is the 3+ charge state of the tryptic peptide TYFPHFNLSHGSDQVK, and the m/z 638.0 ion is the 2+ charge state of LLVVYPWTQR. The CV values in these two IS-CV plots illustrate that the highfield ion mobility characteristics (Kh/K) of these two ions are very different. Figure 8 shows ESI-FAIMS-MS mass spectra collected at the peak maxima of the IS-CV scans displayed in Figure 7 (i.e.,

-7.2 and -10.2 V). These mass spectra are composed of two independent sets of ions. Ions that appear at CV ) -7.2 V (Figure 8a) include m/z 638.0 as well as the peptides KVLQSFSDGLK2+ (m/z 611.4) and VNVDEVGGEALGR2+ (m/z 658.0). All of the ions appearing in Figure 8a (i.e., CV ) -7.2 V) are transmitted with low efficiency at CV ) -10.2 V (Figure 8b) and therefore are at very low intensity in this spectrum. The mass spectrum at CV ) -10.2 V is dominated by the m/z 626.4 ion, but includes lower abundance ions at m/z 590 and 614.5. The m/z 590 ion (tentatively identified as LRVDPVNFKLLSHCLLVTLAHHPD-

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Figure 7. Ion-selected CV spectra for peptide ions m/z 626.4 and 638.0 from a 7.5 µg/mL trypic digest of pig hemoglobin. FAIMS was operated at a DV of -3900 V and the CV was scanned from 0 to -16 V.

Figure 8. ESI-FAIMS-MS mass spectra of a 7.5 µg/mL tryptic digest of pig hemoglobin collected at a DV of -3900 V and (a) a CV of -7.2 V and (b) a CV of -10.2 V. These CV values correspond to the peak maxima in the IS-CV scans for m/z 626.4 and 638.0 shown in Figure 7.

DFNPSVHASLDK7+) cannot be seen in the conventional ESI-MS spectrum shown in Figure 4a. Identification of Isobaric Interferences. Isobaric overlap of ions can pose a significant problem in ESI-MS spectra of complex mixtures. The tryptic digest of pig hemoglobin produced relatively simple mass spectra with few cases of isobaric interferences. Nevertheless, an unresolved overlap between two ions with low relative intensity, AAWGK+ and [MFLGFPTTK + Na]2+ (m/z 532.4), was observed. Figure 9 shows an IS-CV scan for the m/z 532.4 ion. This trace shows that there are two distinct types of m/z 532.4 ions, having optimum transmissions near CV ) -3.2 V and CV ) -8.0 V. Figure 10 shows the mass spectra collected for these two peaks using a separate experiment (DV ) -3850), with data acquired using longer dwell times (50 ms) to improve the clarity of the isotope patterns. The ion transmitted at CV ) -2.7 V (Figure 10a) is the singly charged tryptic peptide AAWGK. On the other hand, the isotope pattern for the ion transmitted at CV ) -6.7 V (Figure 10b) indicates that this ion is doubly charged. The protonated, doubly charged m/z 532.4 ion did not correspond to an ion expected for tryptic cleavage of pig hemoglobin. A series of MS/ MS experiments on the FAIMS-separated m/z 532.4 ion confirmed

Figure 9. Ion-selected CV spectrum for the peptide ion m/z 532.4 from a 7.5 µg/mL tryptic digest of pig hemoglobin. FAIMS was operated at a DV of -3900 V, CV scanned from 0 to -16 V.

Figure 10. ESI-FAIMS-MS mass spectra, using a 7.5 µg/mL tryptic digest of pig hemoglobin, collected for the two peaks detected in the IS-CV scan for m/z 532.4 shown in Figure 9: (a) CV ) -2.7 V; DV ) -3850; 50-ms dwell time per 0.1 m/z (b) CV ) -6.7; DV ) -3850; 50-ms dwell time per 0.1 m/z.

this ion as the sodium-containing peptide [MFLGFPTTK + Na]2+. The doubly protonated MFLGFPTTK ion at m/z 521.4 is prominent in the ESI-MS spectrum shown in Figure 3a and might, therefore, be expected to yield a low-intensity sodiated ion. Inspection of the conventional ESI-MS spectrum (Figure 3a) near m/z 532.4 indicates that doubt about the identity of this ion will occur because the charge state cannot be determined from the isotopic distribution. This problem of unresolved overlap of isobars AAWGK+ and [MFLGFPTTK + Na]2+ was identified, and overcome, using ESI-FAIMS-MS. Improvement in Signal to Background: Why Did the Background Decrease? The detection of low concentrations of peptide fragments using ESI-MS is ultimately limited by the intense chemical background noise that is characteristic of the ESI source. Throughout this document, the FAIMS device is shown to improve the signal-to-background ratio. This section considers some of the reasons for the apparent improvement in the signal-to-background ratio. Three IS-CV spectra are shown in Figure 11. The IS-CV scan for m/z 532.4 ( 0.5 (average of ion intensity from m/z 531.9 to Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

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Figure 11. Ion-selected CV spectra for mass ranges m/z 527.0 ( 1, m/z 532.4 ( 0.5 and m/z 536 ( 1 from a 7.5 µg/mL trypic digest of pig hemoglobin. FAIMS was operated at a DV of -3900 V, and the CV was scanned from 0 to -16 V.

532.9) represents the analyte ion, whereas the two traces at lower (m/z 527.0 ( 1.0) and higher (m/z 536.0 ( 1.0) m/z values monitor a range of background ions. The IS-CV scans of both of the background ions show wide peaks with ion transmission from approximately CV ) -4 to -14 V with maxima near CV ) -9 V. The IS-CV spectrum of m/z 532.4 ( 0.5 shows two maxima at CV ) -3.2 and -8.0 V. From the figure, the mass spectrum of the ion transmitted near a CV of -3.2 V is expected to show very low intensity of background ions, since the most intense background appears near a CV of -9 V. The mass spectra of the m/z 532.4 ions have been discussed above, and the low noise of the spectrum acquired at CV ) -2.7 V is apparent from Figure 10a. For the spectrum collected at CV ) -6.7 V (Figure 10b), the background is much more intense, as would be predicted from Figure 11. Thus, although the intensity of the two ions with m/z 532.4 is comparable in Figure 10a and b, the S/B is greatly improved for the ions appearing in Figure 10a. The FAIMS reduction of the chemical background from ESI is attributed to the following phenomena. A comparison of the CV plots of the analyte and background ions suggests that, in some instances, significant improvements in signal-to-background are a result of the difference in the optimum CV of transmission of the analyte and background ions of the same m/z (described above). Nevertheless, in some cases, the transmission of the analyte will overlap, in part, with the transmission of the background ions. Even when this overlap occurs, many of the ions that would contribute to the background in an ESI-MS spectrum are not transmitted at exactly the same CV of the analyte ion. This simple, low-resolution separation also appears to result in an improvement in the signal-to-background ratio. Finally, the mass spectra collected at all of the CVs investigated in this experiment were added together to yield one spectrum. This “summed” spectrum might be expected to contain all of the contributions to the chemical background and therefore would have a chemical background comparable with the original ESIMS experiment. This is not observed. The background level in this summed spectrum remains lower than that in the equivalent ESI-MS scan. We speculate that many of the ions contributing to the “background” are dynamically changing within the time frame of ion transmission through FAIMS (0.05-0.5 s) and thus are not observed in the final spectrum. Any ion that undergoes a dissociation resulting in a product ion that has mobility characteristics at high electric fields (Kh/K) that are dissimilar to the parent ion 4582 Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

will be lost to the walls of the FAIMS device. For illustration (hypothetical), assume that an ion weakly bound to acetate is transmitted at CV ) -6 V and that the acetate-free ion is transmitted at CV ) -3 V. If the acetate-containing ion is converted to its acetate-free version via loss of acetic acid while the FAIMS is operating at CV ) -6 V, a rapid loss of this product ion will occur since it is transmitted at CV ) -3 V. Similarly, if the FAIMS is operating at CV ) -3 V, formation of the acetate complex will mean that this ion, normally transmitted at CV ) -6 V, will be lost. Peak Shapes in the IS-CV Spectra using FAIMS-MS. Three identifiable types of peak shapes were observed in the IS-CV spectra of the peptides in the tryptic digest of pig hemoglobin. The IS-CV spectra of many ions show peaks that are much narrower than others and have well-defined peak maxima. The IS-CV spectrum of the m/z 638.0 ion appearing in Figure 7 shows an example of a “narrow” peak. A second peak shape is illustrated by the m/z 626.4 ion (also shown in Figure 7). This peak shape is similar to the m/z 638.0 ion in that it also has sharp sides; however, it has a wider maximum. For the peptide ions transmitted at the most negative CV values, ion focusing is strongest and the appearance of “flat-topped” peaks might be expected.12 Finally, the third type of peak shape is similar in shape to that shown in the IS-CV plot of m/z 532.4 shown in Figure 11. Two examples of these wide peaks include m/z 470.2 and 475.0 (CV spectra not shown). The m/z 470.2 ion is transmitted from about CV ) -9 to -15 V, and the m/z 475.0 is transmitted from CV ) -7 to -12 V. The mass spectra collected at the extremes of lower CV and higher CV of these peaks failed to provide any evidence of the presence of multiple overlapping ions. This data suggests that, in some cases, there exists a mechanism by which an analyte ion may be transmitted over a wide range of CV. Three explanations for the unusually wide CV spectra of certain ions may be feasible. First, in some instances the resolution of the FAIMS device may not be sufficiently high to separate a series of similar and/or related ions. A higher resolution FAIMS device may be able to indicate the presence of multiple species (such as adducts), even if these species are each converted to [M + nH]n+ ions during expansion into the mass spectrometer. Second, the ions may exist as several structural conformers (either stable or subject to dynamic changes) that are not separated by FAIMS. This is known to be the case for ions with high mass and low charge (e.g., proteins18); however, it is not known if smaller ions have multiple conformers differing significantly in Kh/K. Finally, the ions may be undergoing dynamic chemical transformations or gas-phase association reactions that tend to yield wide CV spectra. The transmission of ions that are undergoing dynamic chemical changes will be observed if (1) the parent and the product are transmitted at substantially the same CV or (2) the dynamic chemical change is linked to the velocity of the ion during application of the asymmetric waveform. In the second instance, the association and dissociation of weakly bound complexes may be a consequence of the high and low velocity of the ion during the high-field and low-field portions of the asymmetric waveform. Association of the ion with gas-phase neutrals will be preferred during the low-field (low effective temperature, low ion velocity) portions of the waveform, while dissociation of these weak complexes will be preferred during

Table 1. CV and Collision Cross Sections of Ions from a Tryptic Digest of Pig Hemoglobin

a

m/za

charge

massb

CV

identity

266.8 270.8 319.1 338.3 352.4 359.3 370.3 384.1 408.1 409.9 448.5 456.9 470.2 475.0 487.0 521.4 523.4 532.4 544.5 547.5 560.2 563.1 575.4 611.4 614.5 626.4 633.5 638.0 640.5 658.0 682.8 684.7 703.6 712.0 717.5 746.6 758.6 1023.7

2 2 1 1 2 2 2 3 3 2 5 2 4 3 2 2 1 1 2 2 4 3 2 2 3 3 2 2 2 2 3 3 1 2 1 3 3 2

532.3 540.3 319.1 338.2 703.4 717.4 739.4 1149.8 1221.7 818.4 2237.2 912.5 1876.9 1422.7 972.6 1041.5 523.3 532.3 1087.6 1093.6 2237.2 1685.8 1149.8 1221.7 1841.9 1876.9 1265.8 1274.7 1279.7 1314.7 2045.9 2051.9 703.4 1422.7 717.4 2237.2 2273.1 2045.9

-4.5 -3.5 -4.0 -4.0 -5.8 -6.2 -7.0 -8.8 -10.0 -7.4 -9.0 -7.2 -13.2 -8.5 -7.8 -7.2 -3.8 -3.2 -9.4 -7.0 -9.4 -10.0 -7.4 -6.9 -9.5 -10.2 -7.0 -7.1 -7.2 -6.9 -8.8 -10.0 -3.8 -6.8 -4.0 -8.5 -9.2 -6.5

AAWGK AHGKK or AHGQK YH YR VLSAADK VADALTK HLDNLK VVAGVANALAHK KVLQSFSDGLK VDPVNFK AVGHLDDLPGALSALSDLHAHK VHLSAEEK TYFPHFNLSHGSDQVK VGGAGAHGAEALER EAVLGLWGK MFLGFPTTK GTFAK AAWGK LRVDPVNFK VLQSFSDGLK AVGHLDDLPGALSALSDLHAHK LGHDFNPNVQAAFQK VVAGVANALAHK KVLQSFSDGLK RLGHDFNPNVQAAFQK TYFPHFNLSHGSDQVK LLGNVIVVVLAR LLVVYPWTQR FLANVSTVLTSK VNVDEVGGEALGR FFESFGDLSNADAVMGNPK LSELHCDQLHVDPENFR VLSAADK VGGQAGAHGAEALER VADALTK AVGHLDDLPGALSALSDLHAHK FFESFGDLSNADAVMGNPKVK FFESFGDLSNADAVMGNPK

cross section (ref 21)

105.75 110.63 212.8 203.64 222.58 232.31 248.32 153.97 157.36 261.06 255.01 272.31 290.65

287.97 287.78 287.12 193.03 300.48 194.69 377.85

Experimentally observed values. b Theoretical mass for the protonated peptide (monoisotopic).

application of the high fields. This phenomenon of dynamic chemical change is expected to shift the observed CV of the ions, and in some cases, to give peak broadening in the CV spectrum. CV of Identified Tryptic Peptide Ions. Table 1 summarizes the CV of optimum transmission of several of the peptide ions observed in the ESI-FAIMS-MS spectra of a 7.5 µg/mL solution of a tryptic digest of pig hemoglobin. The CV values were determined by extracting IS-CV scans for several of the ions that were apparent in the ESI-FAIMS-MS spectra. Note that this is not an exhaustive list of the fragment ions discernible in the ESIFAIMS-MS spectra. The data recorded in Table 1 permits a qualitative comparison of the high-field behavior (Kh/K) of these ions under one set of experimental conditions. The singly charged ions reported in Table 1 are transmitted through FAIMS at a compensation voltage of about -4.0 V. Most of the doubly charged ions are transmitted between CV ) -6 and -8 V. The set of 10 triply charged ions are transmitted between CV ) -8.5 and -10 V. The higher charged ions are transmitted at CVs up to -13.2 V. This general separation of ions as a function of charge state suggests a practical method for rapid collection of ESI-FAIMS data by selection of a limited number of measurements in the CV range of interest. Since most of the multiply charged ions are transmitted above CV ) -6 V, and each ion is usually transmitted over a CV range of at

least 2 V (see Figure 7), data could be acquired in CV increments of ∼0.5 V. Therefore, in practice, a majority of the useful information about the multiply charged ions in this tryptic digest could be collected by a series of eight measurements ranging from CV ) -6 to -10 V. The separation of the multiply charged ions from most of the singly charged ions offers practical advantages for MS/MS experiments. Spectra lack many of the interfering ions typically observed in the conventional ESI-MS/MS spectra of multiply charged peptides when the background of singly charged ions is intense relative to the parent ion. The MS/MS spectra thus acquired will be less difficult to interpret for the elucidation of the peptide sequence. The experimental value of CV is dependent on many parameters, including the following: DV, the shape of the applied asymmetric waveform, the width of the FAIMS analyzer region, the gas pressure and temperature in FAIMS, the type of gas, and the functional behavior of the high-field mobility of the ion. Consequently, the CV values for the peptides shown in Table 1 are for a specific set of experimental conditions. A more generalized description of the high-field behavior of the (Kh/K) for each ion would provide the basis for calculation of the expected CV for any particular experimental condition. A complex experimental and data processing procedure, described elsewhere,20 is required Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

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Figure 12. The CV of optimum transmission of several peptides from a 7.5 µg/mL tryptic digest of pig hemoglobin as a function of (a) massto-charge ratio, (b) total peptide mass, (c) peptide ionic charge, and (d) peptide collision cross section (ref 21).

to convert the raw data of CV and DV into a quantitative description of the high-field behavior of the ion. Fortunately, this conversion of raw CV and DV data to a quantitative description of (Kh/K) as a function of electric field is not essential for the practical application of FAIMS. The Relationship between CV, Mass-to-Charge Ratio, and Total Ion Mass. Figure 12a shows a plot of the CV of the optimum transmission of several of the ions identified in the tryptic digest of pig hemoglobin (i.e., from Table 1) as a function of m/z. This plot shows that virtually no relationship exists between the mass-to-charge ratio and the CV observed for these ions. In conventional drift tube IMS experiments, the reduced mobility (K0) of an ion is related to ionic charge and collision cross section.14 In general, this automatically leads to a relationship between m/z and the mobility of an ion, as shown in plots of the tryptic fragments collected by drift tube ion mobility.21 Also included in Table 1 are the measured collision cross sections21 of a limited number of the singly and doubly charged peptides. Figure 12b shows a plot of the CV of maximum transmission of the tryptic digest peptide ions as a function of the total mass of the peptide. The correlation between the observed CV and the mass of the peptide is significantly better than the correlation between CV and m/z. Figure 12c shows a similar plot of CV as a function of charge state for the same peptide ions. Again, a limited correlation exists between the measured CV and the ionic charge for this narrow range of charge states. Figure 12d shows a plot of ion collision cross section21 as a function of CV for a limited selection of ions with +1 and +2 charge states (see Table 1). The ions with charge state +1 are all (21) Valentine, S. J.; Counterman, A. E.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1999, 10, 1188-211.

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transmitted at a CV near -4 V, whereas those ions at +2 appear near a CV of -7.5 V, with the exception of LRVDPVNFK which appears near a CV of -9.4 V. These limited results suggest that separations in FAIMS are orthogonal to those in conventional drifttube IMS experiments, since the ions that appear at very similar CV values in FAIMS may be separated in an IMS experiment. A tandem combination of IMS and FAIMS might be expected to offer better separations than either FAIMS or IMS alone. CONCLUSIONS A new design of FAIMS has been evaluated for applications involving the mass spectrometric analysis of tryptic digests of proteins. The use of an inner electrode that terminates in a hemispherical shape is shown to bring the ions, via the gas flow, to the center axis of the electrode and provide improved ion transmission into the mass spectrometer. The continuous ion separation and focusing abilities of FAIMS dramatically improves the signal-to-background ratio for tryptic fragments of pig hemoglobin thereby significantly lowering the detection limits relative to conventional ESI-MS. For the tryptic peptides of pig hemoglobin, a correlation (1) between CV and the total mass of the ion and (2) between CV and the ionic charge state was observed. However, the correlation between CV and the mass-to-charge ratio of the ion was almost nonexistent. This low correlation makes FAIMS an ideal gas-phase processing tool for separation of electrospray-generated ions prior to their introduction into a mass spectrometer. Received for review January 5, 2000. Accepted July 7, 2000. AC0000271