Investigation of the Rapid Scan on an Electrospray ... - ACS Publications

A rapid scan on a quadrupole ion trap mass spectrometer can improve the signal intensity by over 200 times when scanning at 12 times the normal scan r...
0 downloads 0 Views 334KB Size
Download PDF
Anal. Chem. 2005, 77, 1663-1671

Investigation of the Rapid Scan on an Electrospray Ion Trap Mass Spectrometer Chunguang G. Yang and Mark E. Bier*

Center for Molecular Analysis, Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213

A rapid scan on a quadrupole ion trap mass spectrometer can improve the signal intensity by over 200 times when scanning at 12 times the normal scan rate. This intensity increase is due to a 5.5-fold increase in mass peak height due to a reduction in the mass peak width over time and a 40-fold increase in signal from the increased number of ions that can be trapped without the deleterious effects of space charge. Detection limits can be further improved by signal averaging more scans in the same period that is required for the normal scan, and the greatest advantage occurs when scanning over the full mass range. The rapid scan impacts the mass accuracy and the resolution is reduced by 6 times. The molecular weight determination of 40 fmol/µL apomyoglobin was determined in 3 s using a rapid scan, but this was not possible when using the normal scan rate. Quantitation results showed that the relative standard deviations for the total ion current peak areas of 500 fmol of angiotensin I were improved by a factor of 2.6 when the rapid scan was used. Quadrupole ion trap mass spectrometers have been widely used as powerful and feature-rich analytical instruments.1-3 The ion trap analyzer has been coupled to both GC4,5 and LC6-9 instruments and also to other analyzers such as magnetic sector instruments and FTMS.10 An assortment of scan modes has been developed for ion traps including full scan MS,11 MSn,12,13 selected * To whom correspondence should be addressed. Phone: 412-269-3540. Fax: 412-268-6897. E-mail: [email protected]. (1) March, R. E. J. Mass Spectrom. 1997, 32, 351-369. (2) March, R. E. Rapid Commun. Mass Spectrom. 1998, 12, 1543-1554. (3) Cooks, R. G.; Kaiser, R. E., Jr. Acc. Chem. Res. 1990, 23, 213-219. (4) Larsson, L.; Saraf, A. Mol. Biotechnol. 1997, 7, 279-287. (5) Sablier, M.; Fujii, T. Handb. Spectrosc. 2003, 2, 244-278. (6) Creaser, C. S.; Stygall, J. W. TrAC, Trends Anal. Chem. 1998, 17, 583593. (7) Niessen, W. M. A. J. Chromatogr., A 1998, 794, 407-435. (8) Kleintop, B. L.; Eades, D. M.; Jones, J. M.; Yost, R. A. Pract. Aspects Ion Trap Mass Spectrom 1995, 3, 187-214. (9) Niessen, W. M. A.; Tjaden, U. R.; Van der Greef, J. Methodol. Surv. Biochem. Anal. 1992, 22, 253-260. (10) Murrell, J.; Konn, D. O.; Underwood, N. J.; Despeyroux, D. Int. J. Mass Spectrom. 2003, 227, 223-234. (11) Stafford, G. C., Jr.; Kelley, P. E.; Syka, J. E. P.; Reynolds, W. E.; Todd, J. F. J. Int. J. Mass Spectrom. Ion Processes 1984, 60, 85-98. (12) Louris, J. N.; Cooks, R. G.; Syka, J. E. P.; Kelley, P. E.; Stafford, G. C., Jr.; Todd, J. F. J. Anal. Chem. 1987, 59, 1677-1685. (13) McLuckey, S. A.; Glish, G. L.; Kelley, P. E. Anal. Chem. 1987, 59, 16701674. 10.1021/ac048636m CCC: $30.25 Published on Web 02/11/2005

© 2005 American Chemical Society

ion monitoring (SIM),14 SRM,15 and high resolution.16 In 1995, a scan mode was introduced based on increasing the scan rate.17 The scan rate is defined here as the rate at which ions are scanned out of the ion trap, (m/z)/s, during the mass analysis segment of the scan. This scan mode shortened the overall scan time, increased signal heights, and improved detection limits. From the preliminary results presented in that early report it was suggested that this rapid scan was analytically useful, especially in the full scan mode where the analytical scan period can dominate the total scan time. Few references to the rapid scan have been found in the literature18 despite the fact that it was eventually added as a scan feature to a commercial ion trap mass spectrometer. One of the reasons for the underutilization of this scan mode is perhaps due to the lack of understanding of the advantages of using the rapid scan. We have studied the rapid scan mode extensively and discuss the advantages and limitations in this article. In the 1980s, the scan rate was first increased during the development of the prescan to allow for variable ionization times or what was later called automatic gain control (AGC).19 The AGC prescan is used to measure the ion signal and then adjust the ion injection time to optimize the number of ions to be trapped for the analytical scan. If too few ions are trapped, poor signal-tonoise (S/N) ratio results, while if too many ions are trapped, broad peaks and a mass shift results due to the effects of space charge. The scan rate was increased for the AGC prescan because a rapid means of measuring the ion signal was required to maintain the high ion trap duty cycle. To speed up the prescan, the slope of the rf amplitude in (volts/second) for AGC mass analysis was increased. In 1989, the mass range of the ion trap was extended by Kaiser and co-workers20,21 by resonantly ejecting ions at a lower qeject while (14) Creaser, C. S.; Mitchell, D. S.; O’Neill, K. E. Int. J. Mass Spectrom. Ion Processes 1991, 106, 21-31. (15) Todd, J. F. J. Pract. Aspects Ion Trap Mass Spectrom. 1995, 1, 3-24. (16) Schwartz, J. C.; Syka, J. E. P.; Jardine, I. J. Am. Soc. Mass Spectrom. 1991, 2, 198-204. (17) Bier, M. E.; Schwartz, J. C.; Zhou, J.; Syka, J. E. P.; Taylor, D.; Land, a. P.; James, M.; Fies, B. 43rd ASMS Conference on Mass Spectrometry and Allied Topics, 1995; p 988. (18) Ogueta, S.; Rogado, R.; Marina, A.; Moreno, F.; Redondo, J. M.; Vazquez, J. J. Mass Spectrom. 2000, 35, 556-565. (19) Stafford, G. C., Jr.; Taylor, D. M.; Bradshaw, S. C., Method of increasing the dynamic range and sensitivity of a quadrupole ion trap mass spectrometer. U.S. Patent 5,107,109, 1992 (20) Kaiser, R. E., Jr.; Louris, J. N.; Amy, J. W.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1989, 3, 225-229. (21) Kaiser, R. E., Jr.; Cooks, R. G.; Moss, J.; Hemberger, P. H. Rapid Commun. Mass Spectrom. 1989, 3, 50-53.

Analytical Chemistry, Vol. 77, No. 6, March 15, 2005 1663

maintaining the same rate of increase of the rf amplitude (volts/ second). By lowering the qeject resonance point in their experiments, they increased the mass range and scan rate ((m/z)/ second) without increasing the main rf amplitude on the ring electrode. For example, since the maximum mass is inversely proportional to qeject, by lowering the resonance ejection point from qeject ) 0.85 to qeject ) 0.085, the mass range and scan rate can be increased by 10-fold. The objective of Kaiser’s experiment was to extend the mass range, so no extensive study to characterize the peak shape, signal improvement, or detection limit was pursued. This study is focused on the use of a rapid scan to examine how it affects the mass accuracy, resolution, detection limits, and quantitation capabilities of the instrument. We suggest and demonstrate the best applications for the rapid scan and note how the implementation of this scan can be improved so that it can be effectively applied to real analyses. EXPERIMENTAL SECTION Materials. Angiotensin I (human) was purchased from American Peptide Co. (Sunnyvale, CA). Apomyoglobin was obtained from Sigma Chemical Co. (St. Louis, MO). Samples were made into stock solutions of 1000 pmol/µL and stored in a freezer in aliquots of 100 µL. One aliquot was taken out and diluted to the desired concentration for use in the rapid scan experiments. The CsI solution was made by dissolving 50 mg of CsI in 20 mL of acetonitrile and infusing it directly. All solvents were HPLC grade and purchased from Fisher Scientific (Fair Lawn, NJ). Ion Trap Mass Spectrometer. The electrospray ion trap mass spectrometer used in this study was a LCQ model from Thermo Electron Corp. (San Jose, CA). The software used on the LCQ was Xcalibur version 1.1. This software included a mass deconvolution algorithm to determine the molecular weight from a spectrum of multiply charged ions. Although the name Turboscan was adopted by Thermo Electron Corp., we use the term “rapid scan”, which is more descriptive of this scan. The scan rate for the LCQ ion trap mass spectrometer was set to either the normal rate of 5555 (m/z)/s or the rapid rate of 12× the normal scan rate, 66 660 (m/z)/s. Because we did not have access to the software necessary to investigate and compare a variety of different scan rates, we only investigated the 1× and 12× the normal scan rates. Helium was maintained in the ion trap at ∼1 mTorr. Methods. A Tektronix TDS 520D oscilloscope (Wilsonville, OR) with a maximum digitization rate of 2 gigasamples (GS)/s was used to make more precise and accurate waveform measurements than what was possible using the LCQ data system. Oscilloscope measurements of a mass peak were performed by averaging 200 waveforms at a sampling rate of 5 MS/s during the direct infusion experiments unless noted otherwise. The ion signal was measured at TP3 on the analyzer board. The main rf scan function was measured at pin 1 (mod_in) of TP12 on the analyzer board. The trigger signal was measured at pin 3 of TP15 on the system board. The trigger was set to the beginning of each scan by choosing “scanout” on the “Triggering” page in “Diagnostics” dialogue box from the “LCQ tune” program. The data were saved in proprietary Tektronix waveform (WFM) format and extracted with CNVRTWFM version 1.97 supplied by Tektronix. Statistical analyses were done using Origin 6.1 by OriginLab Corp. (Northampton, MA). Rapid scanning was enabled by selecting the “Turboscan” in the “Define Scan” window. For some experiments, 1664 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

Figure 1. Oscilloscope traces of (a) a normal scan function, (b) a rapid scan function, and (c) the trigger signal for the normal microscan. The mass range was from m/z 600 to 1200. The ion injection time was 20 ms, and oscilloscope data sampling rate was 250 kS/s. In this example, three rapid scans can be acquired in the same time it takes to collect one normal scan.

the AGC, which automatically sets the ion injection time based on a prescan measurement, was turned off and the ion injection time was set manually. The definition of space charge in mass spectrometry refers to the perturbation or distortion of an electric field by one or more point sources (ions). For example, by having one or more ions placed in a quadrupole field, the quadrupole field becomes distorted from ideal. Signs of the effects of space charge in ion trap mass spectrometry include broadening and merging together of mass peaks and the shifting of the mass assignment higher. In our study, we defined the evidence for the onset of space charge effects as a permanent mass shift,22,23 where ∆ m/z is +0.1 above the mass accuracy error for which the mass spectrometer is specified. For quantitation experiments, flow injection analysis (FIA) was performed by coupling a MAGIC 2002 HPLC system from Michrom BioResources Inc. (Auburn, CA) with the LCQ. Mobile phase A was 99.9% water and 0.1% acetic acid, and mobile phase B was 99.9% acetonitrile and 0.1% acetic acid (v/v). The final mobile-phase concentration was 50% A and 50% B. The injection loop used had a volume of 5 µL, and it was connected directly to the electrospray ionization source using a fused-silica capillary tube with a 100-µm internal diameter. The LC flow rate was set to 30 µL/min. The sheath gas flow was set to 60 (arbitrary units), and the auxiliary gas flow was set to 20 (arbitrary units). RESULT AND DISCUSSION The digital oscilloscope recordings of the rf amplitude for the normal and rapid scan functions at 1× and 12× the normal scan rate, and the trigger signal for the start of the normal scan are shown in parts a-c of Figure 1, respectively. The trigger signal waveform marks the beginning and end of a normal scan over a (22) Todd, J. F. J.; Waldren, R. M.; Mather, R. E. Int. J. Mass Spectrom. Ion Phys. 1980, 34, 325-349. (23) Guan, S.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1994, 5, 64-71.

mass range from m/z 600 to 1200. The ion injection time was set to 20 ms for both scans and is shown segmented at two different injection rf levels. The mass analysis segment of the two scan functions is labeled, and the rate is shown to be 12× faster in Figure 1b compared to 1a. We used the digital oscilloscope to measure the expected scan rates to within 1% of the expected 5555 (m/z)/s for the normal scan and 66 660 (m/z)/s for the rapid scan. The approximate rate of increase of the rf amplitude for the high-resolution, normal, and rapid scans are 1040, 20 800, and 250 000 V/s, respectively. All scan modes are supplemented by a resonance ejection waveform that is simultaneously applied to the two end caps in dipolar fashion during the scanout. The resonance ejection amplitude is optimized to maximize the resolution, and to do so, these amplitudes are increased with increasing scan rate and m/z with typical values of 1.04, 2.78, and 190 V/s for the highresolution, normal, and rapid scans, respectively. As shown in Figure 1, by increasing the slope of the rf amplitude, the resulting scan function is shortened by such an extent that three rapid scans can be implemented in the same period of time that it takes to complete one normal scan over a mass range of ∆m/z 600. As can be determined from Figure 1, it would be possible to collect nine rapid scans in the time period it would take to collect one normal scan over the entire mass range m/z 50-2000 and this would increase the signal-to-noise level by n1/2 ) 3 (where n is equal to the number of rapid scans that can be collected in the equivalent normal scan period). The S/N ratio improvement is reduced at higher ion injection times where the duty cycle is reduced, but for an experiment at ion injection times of 100 ms and a mass range of m/z 50-2000, only one normal scan (0.98 s) can be acquired in the time it takes to collect five rapid scans (1.09 s). In this example, the duty cycle is 45 and 10% for the rapid and normal scans, respectively. Ion Signal at the Normal and Rapid Scan Rate. Although the total number of ions trapped in the analyzer should be the same for the normal and rapid scans provided that the ion injection time is the same, it was not clear whether all of these ions would be ejected from the ion trap equally at 1× and 12× the normal scan rate at various ion abundances. To explore the efficiency of ejection at these two different scan rates, we collected the ion signal from doubly charged angiotensin I at m/z 648.8 for [M + 2H]2+. To select only the doubly charged ion isotope distribution, we operated the mass spectrometer in the SIM mode with an isolation window of ∆m/z 10. The narrow isolation window reduced the possibility of chemical interference. Using the digital oscilloscope, we collected the peak areas at the normal and rapid scan rates and plotted these against various ion injection times in Figure 2. As can be seen in Figure 2, the total peak areas versus ion injection times were the same at ion injection times less than 10 ms, but at greater times, the rapid and normal scans show a 10 and 20% negative deviation, respectively, compared to the expected ion abundance from an extrapolation of the first five data points of the rapid curve (dashed line). In this experiment, part of this reduction may be due to a loss in ion injection or trapping efficiency at the higher ion abundances, which should be the same for both scan modes, and a decrease in ion ejection efficiency when using a slower scan rate due to space charge effects. In addition, collisions with helium at 1 mTorr may further disrupt a slow ejection process by ion scattering. What is noteworthy from

Figure 2. Peak area of the doubly charged angiotensin I [M + 2H]2+ versus ion injection time collected at 1× (4) and 12× (0) the normal scan rate. The LCQ was set to SIM mode with the center mass set at m/z 649 and with an isolation width of a ∆m/z of 10. The ion injection time was manually set, and the oscilloscope was set to a data sampling rate of 5 MS/s. The rapid and normal scan peak areas are similar at short ion injection times, but a slight negative deviation is observed at long ion injection times.

Figure 3. Height of a mass peak from angiotensin I, [M + 2H]2+, versus the ion injection time. The plot with (O) is the linear fit of the rapid scan data, and the plot with (4) is the normal scan rate data. The LCQ was set to SIM mode with a 10 ∆m/z isolation mass window centered at m/z 649. The ion injection time was set manually, and the oscilloscope was set to a sampling rate of 5 MS/s. The rapid scan peak heights are 5.5 times greater than the peaks acquired by the normal scan rate.

these data is that the number of ions trapped and then detected is essentially the same at these different scan rates. Injecting the same number of ions in the trap and scanning them out in a shorter period not only reduces the time of analysis but also significantly increases the mass peak signal height. Peak heights were measured for doubly charged angiotensin I at a concentration of 50 pmol/µL in SIM mode for various ion injection times and plotted in Figure 3. The slope of peak heights for the rapid scan data versus ion injection time was found to be 5.5 times greater than the normal scan. The inset shows that at ion injection times greater than 1.5 ms (thicker line segment) the normal scan rate spectrum was determined to be space charged. The exact Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

1665

Figure 4. Mass spectra of doubly charged angiotensin I [M + 2H]+2 over a 10 ∆m/z SIM window for (a) a normal scan with an ion injection time of 0.5 ms, (b) a rapid scan with an ion injection time of 0.5 ms, (c) a normal scan with an ion injection time of 2 ms, and (d) a rapid scan with an ion injection time of 12 ms. A loss of resolution is seen at the higher ion injection time for the normal scan while the rapid scan has no resolution loss and an increase in peak height. The data were collected and exported from Xcalibur and redrawn using Origin.

ion injection time for this mass shift at longer times for the rapid scan, however, was not possible in this experiment because the preamp on the analyzer board became saturated producing a flattopped peak. Despite this problem, remarkably, ∼40× more ions can be trapped when the rapid scan mode is used without the onset of space charge producing an effective sensitivity gain of 40. Of course, chemical noise due to nonanalyte peaks will also increase by an equal amount. A number of features of the doubly charge angiotensin I peaks are lost in the rapid scanning mode, but the significant gain in ion signal makes the rapid scan mode advantageous. Mass peaks collected in the SIM mode using the LCQ data system at 1× and 12× the normal scan rate are shown in Figure 4. Figure 4a shows that, at an ion injection time of 0.5 ms, the first two doubly charged isotope peaks were resolved with a 60% valley. At the rapid scan rate, the isotope peaks were unresolved in Figure 4b, but the peak height was significantly increased. As shown in Figure 4c, at 2-ms ion injection time, the doubly charged peak height increased by 4 times, but the isotope peaks are no longer well resolved and the mass is shifted higher due to the effects of space charge. In contrast, the data in Figure 4d were collected at an ion injection time by 24 times greater than Figure 4b with no change in resolution or mass assignment, but with an increase in peak height of ∼24 times. The rapid scan data show the improved signal height and the increase in the number of ions that can be trapped and scanned out of the ion trap with no deleterious space charge effects. It should be pointed out that the ion peak shapes in the rapid scan spectra in Figure 4b and d are not well-defined by a sufficient number of points using the LCQ data system. The limitations of this digitization rate will be discussed in a later section. Rapid Scan Resolution. As discussed and shown in the data above, the signal height is improved, but the resolution is reduced when using the rapid scan. The normal scan can easily resolve the isotopic peaks up to m/z of 2000 and charge of two on the LCQ, whereas the rapid scan cannot resolve the isotopic peaks regardless of the ion injection time or mass. To determine the 1666 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

intrinsic resolution at all scan modes, we selected a cesium iodide ion cluster20 since cesium and iodine each have only one natural isotope. No attempt was made to change the helium pressure when the mass resolution was determined. In addition to the normal and rapid scan rates, we collected a mass spectrum of the cesium iodide cluster ions in the high-resolution scan mode. We selected Cs4I3+, m/z 912.33, to calculate the resolution using the R ) m/∆m FWHM definition. At this m/z, we determined a resolution of 5066, 1957, and 308 for the high-resolution, normal, and rapid scan modes, respectively. It is interesting to note that the scan rate for the high-resolution scan is 1/20 the normal scan, and this is a low-sensitivity scan due to the reduced number of ions allowed before the onset of space charge effects.16 Since the rapid scan is not capable of resolving the isotopes of angiotensin I, especially for the multiply charged ions, we assigned the centroid of the peak to an average mass rather than a monoisotopic mass. The isotopes are unresolved in the normal scan mode for multiply charged ions above a charge state of three and so the reduced resolution in the rapid scan mode is less important when large peptides are analyzed since, in either case, we resort to an average mass assignment. Rapid Scan Mass Accuracy. Mass accuracy depends on many parameters, such as the main rf frequency, the resonance ejection frequency, the resonance ejection amplitude, the number of trapped ions, the kinetic energy of the ions, the helium pressure, and the scan rate.24 The mass accuracy of the LCQ is ∼0.01% under typical operating conditions in the normal scan mode.25 As described earlier, at higher scan rates, we can trap more ions than with the normal scan before the onset of space charge effects as defined earlier as a ∆m/z +0.1 shift. We measured the mass shift for both a normal and rapid SIM scan (mass range ∆m/z 300) at various ion injection times for the triply charged ion from angiotensin I. Both the [M + 2H]2+ and [M + 3H]3+ ions were trapped, but only the mass assignment of the triply charged ion at m/z 432.9 was plotted as shown in Figure 5. The solid lines are drawn as a visual aid in this figure. At the normal scan rate, as shown by solid circles (b), the mass assignment exceeds ∆m/z +0.1 at ion injection times above 1 ms and the mass shifts high by ∆m/z +0.3 at 5 ms, which is 5 times above the ion injection time for the defined onset of space charge in this experiment. The mass shift levels off at ∆m/z ∼+1.2 at ion injection times above 30 ms, which appears to be near the limit for the maximum number of trapped ions that can be ejected at these experimental conditions. The rapid scan mass assignments plotted as solid squares (9) does not shows a significant mass shift above ∆m/z +0.1 until an ion injection time of ∼40 ms. Above 40 ms, a further increase is observed in the mass shift slope. It is presumed that, when “space charged”, the trapped ion cloud becomes further dispersed due to ion-ion repulsion that disrupts the focusing ability of the quadrupole field, and as a result, the resolution is reduced. In addition, such a high density of charge results in an effective dc offset of the stability diagram and a mass shift to a later ion ejection time as described by Todd.22 We further investigated the effects of space charge on mass accuracy at both scan rates using a peptide mixture of angiotensin (24) Goeringer, D. E.; Whitten, W. B.; Ramsey, J. M.; McLuckey, S. A.; Glish, G. L. Anal. Chem. 1992, 64, 1434-1439. (25) Bier, M. E. Mod. Protein Chem. 2002, 71-102.

Figure 5. Mass shift of angiotensin I [M + 3H]3+ versus ion injection time. The data were collected using the oscilloscope with a sampling rate of 5 MS/s. In the normal mode (O), the mass values were taken from the top of the resolved monoisotopic peaks if resolved or the average mass if not resolved. Mass assignments for the rapid scanned peaks (9) were determined from the centroid calculated with an in-house program. The mass shifted above ∆m/z +0.1 in the normal scan at ion injection times greater than 1 ms whereas the rapid scan mass peak shifted high by ∆m/z +0.1 above an ion injection time of ∼40 ms. The solid lines are drawn for a visual aid.

I, bradykinin, and ACTH 18-39. Our normal scan data agree with previous experiments showing that the first ions to show signs of space charge with increasing ion injection times are at low m/z.26,27 This occurs because the ejection of ions from the ion trap during mass analysis is from low m/z to high m/z, which means that higher m/z ions are ejected when the trap is at a fraction of the original ion count. This is also true for the rapid scan; however, the effects of space charge are not observed until the ion injection time was increased by more than an order of magnitude higher for the [M + 3H]3+ ion of angiotensin I. This experiment further confirmed the higher space charge tolerance of the rapid scan. After using the rapid scan mode to make several mass measurements, we observed that the mass assignments were always less than the theoretical values. Examination of the calibration parameters used in the LCQ, revealed part of the explanation for this systematic error. The theoretical monoisotopic masses were presumably entered into the LCQ software instead of the theoretical average masses to calibrate the rapid scan (and thus the AGC prescan), but since the isotope mass peaks are not resolved when the rapid scan is used, average mass values should be used to create a more accurate calibration. Although this mass discrepancy is not important for the prescan, which is only used to determine the proper ion injection time for the analytical scan, this mistake does create a considerable mass accuracy error in the rapid scan mode. We did not have access to the software to correct the calibration error; however, the significance of the error is realized by calculating the mass difference between the singly (26) Cleven, C. D.; Cox, K. A.; Cooks, R. G.; Bier, M. E. Rapid Commun. Mass Spectrom. 1994, 8, 451-454. (27) Murphy, J. P., III. Dissertation, Fundamental studies of the quadrupole ion trap mass spectrometer: compound-dependent mass shifts and space charge. University of Florida, Gainesville, FL, 2002.

charged monoisotopic mass and the average mass for each of the five calibration ions. The mass errors for the [M + H]+ ions from caffeine, the tetrapeptide MRFA, and three fluorinate phosphazenes (C24H19O6N3P3F36+, C30H19O6N3P3F48+, and C36H19O6N3P3F60+) are -0.1105, -0.3925, -0.2969, -0.3611, and -0.4253 u, respectively. This error is multiplied when calculating the molecular weight of a protein from ions that are multiply charged. For example, a hypothetical multiply charged protein ion with a molecular weight of 18,286.5, would have 15 bound protons at m/z 1220.1 and an empirical molecular weight less than the theoretical mass by 4.5 u. We determined the theoretical and observed molecular weights for angiotensin I and apomyoglobin at the normal and rapid scan rates. After the correction, we measured a mass accuracy of 0.01% for angiotensin I and 0.03% for the average molecular weight of apomyoglobin using the rapid scan. Even after correcting the calibration error manually, we still found that the m/z values were consistently lower than the theoretical values. We considered that the rf amplitude may not be able to track the rf DAC settings at this rapid scan rate, but a rapid scan calibration is already preformed that should correct for a constant tracking error. Also, a reduced rf amplitude during the analytical scan would shift the m/z assignment higher not lower. A shift in mass due to an insufficient digitization rate should create mass assignment errors, but these errors should average out during the deconvolution calculation. We considered the possibility that there might be an additional systematic error between the scan function used to collect the AGC prescan during calibration and the rapid scan function employed during the mass analysis. In support of this hypothesis was an experiment conducted by triggering from the main rf DAC gain (pin 1, TP12 on the amplifier board) at a specific voltage and measuring the resonance ejection voltage. A lower resonance ejection voltage was measured for the AGC prescan than for the rapid scan. This perhaps explains why the rapid scan peak is detected at a lower rf, but the exact difference between the two scan functions, if any, is left to the manufacturer. In any event, the true mass accuracy is better than what can be measured currently. Peak Shape and the Effects of the Digitization Rate. We examined the impact of the digitization rate used on the LCQ ion trap for the rapid scan by comparing the peak profiles collected using the digital oscilloscope. The true peak shape of a mass peak is not captured by the relatively slow digitizer used on the LCQ. The LCQ records the ion signal with a 16-bit digitizer (Analog Device 776). This ADC is capable of 100 kilosamples (kS)/s with a maximum low band-pass of 45.5 kHz, but on the LCQ, the digitization rate is apparently set to 83 kS/s (based on the points in the mass list) and the low band-pass is expected to be ∼41 kHz. In any regard, the ADC will filter out any of the high frequency components and the resulting spectrum will be effectively smoothed as seen when viewing the peak shape on the LCQ data system. The 83 kS/s digitization rate also means that a 1 m/z interval in the normal scan rate is defined by 16 points (or 15 intervals/(m/z)) while a 1 m/z interval in the rapid scan mode is defined by a mere 1.5 points. To examine the shape of a rapid scan mass peak without the interference of isotope contributions, we acquired a mass spectrum of Cs5I4+ at m/z 1172.14 using the LCQ data system. The mass Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

1667

Figure 6. Oscilloscope traces of Cs4I5+ at m/z 1172.14 from (a) a normal scan and (b) a rapid scan mode. The LCQ was set to the SIM mode with a width of 10 ∆m/z and centered at m/z 1172. The ion injection time was 10 ms. The oscilloscope sampling rate was set at 2.5 MS/s and smoothed using a five-point adjacent averaging method (Origin software). The data were aligned to aid in the peak comparison. Both the secular frequency at 348 kHz and a beat frequency of 64 kHz are observed. The rapid scan peak is narrower than the normal scan peak in time, yet the areas of the two signals are approximately the same.

peak had a width at the base of 6.4 ∆m/z and a FWHM of 3.1 ∆m/z. This broadened peak is defined by 10 data points using the LCQ digitizer. The undersampling for the rapid scan peak would result in a reduced accuracy of the peak top and centroid and thus would result in a reduced mass accuracy. This result can be observed in Figure 4b and d by the angled peak top for the doubly charged unresolved angiotensin I. The fast digital oscilloscope was used to further investigate the true peak shape of both the normal and rapid scan peaks. We chose to examine the peak profile of Cs5I4+, m/z 1172.14, and we used a digitization rate of 2.5 MS/s at full band-pass. These data are shown in Figure 6a and b, where the mass peak of Cs5I4+ is shown acquired at the normal scan rate and 12× the normal scan rate, respectively. The peaks were aligned in time for comparison. Several notable features of these mass peaks are observed. As seen in the figures, the normal and rapid scans have a base peak width of about 200 and 90 µs and a FWHM of approximately 90 and 40 µs, respectively. Here it is clearly evident that although the rapidly scanned mass peak is broader in mass, it is actually narrower in time, and as determined earlier, the peaks are of approximately the same area. It should be noted that although the rapid scan rate is 12× the normal scan rate, the peak width is only ∼2.2 times shorter in time. In addition, the traces show two main frequencies in the profile data for these mass peaks that were determined to be 348 kHz, which is the secular frequency of the ions during ejection, and 64 kHz, which is a beat frequency of 2× the secular frequency, and the main rf frequency of 760 kHz. Approximately 12 beats are seen over the mass peak collected in the normal scan mode versus 6 beats recorded over 1668 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

the peak in the rapid scan mode. The LCQ ADC would filter out this high-frequency content. We also measured the true peak shape of angiotensin I, [M + 2H]2+, at m/z 648.8, and its isotopes as a function of m/z. Figure 7 shows the same frequency content as in the Cs5I4+ spectrum above, but since this is a plot of ion intensity versus m/z and not time, there is a difference between the spacing of the highfrequency signals observed in Figure 7. In Figure 7a, the isotope peaks are resolved at the normal scan rate, but at the rapid scan rate, what can be mistaken as an isotope separation is actually the beat frequency discussed above. The isotopes are unresolved at this fast rate. The periodicity due to the secular motion and the beat frequency clearly does change the peak shape. Accordingly, this beat signal could affect the mass assignment in the rapid scan mode. Applications of the Rapid Scan Mode. Protein and Polymer Molecular Weight Determinations. To demonstrate the utility of the rapid scan, we collected several mass spectra from 40 fmol/µL apomyoglobin (MW ) 16 951) infused at 10 µL/min. Figure 8a shows one 5.6-s normal scan consisting of three averaged microscans. Deconvolution of these data does not result in a molecular weight determination because too few ions make up this mass spectrum. Figure 8c shows one 3.2-s rapid scan of the same apomyoglobin sample at 12× the normal scan rate using the same ion injection time. The signal intensity was improved from 4.94 × 104 to 2.80 × 105 counts, which is an improvement by a factor of 5.6, and a mass deconvolution was possible. Figure 8b is an acquisition of 138 microscans collected at the normal scan rate over 74 s, which is compared to Figure 8d, which shows 741 rapid microscans acquired in a period of 69 s. Although the normal scan molecular weight determination for apomyoglobin was only in error by 1 Da when 138 microscans were collected, the rapid scan allowed for a determination with only three microscans. In addition, six rapid scans can be collected for each normal scan, which allows for a significant S/N ratio improvement as shown in Figure 8d. No mass correction was made to the rapid scan molecular weight determination. What is evident, however, is that the error again appears to be systematic as shown in Figure 8c and d, where the two different sets of data resulted in a precision of 1 Da at 17 kDa. With the correction of the systematic errors discussed earlier, one would expect a mass accuracy of ∼0.01%. This set of figures shows that the rapid scan can be used to give improved detection limits and a greater number of rapid scans can be collected in the same time period for improved signal averaging. The latter improvement would be especially advantageous when doing chromatography where the analysis time is limited. Another example of how the rapid scan mode can be informative as a method of screening or surveying is shown in Figure 9. Here we attempted to analyze a sample for peptides at a low amount, but instead, we found a series of peaks that were identified as a poly(ethylene glycol) (PEG) contaminant. Although no peptide peaks were identified, the PEG peaks visible in the rapid scan spectrum of Figure 9b confirmed that the instrument was working and suggested that the peptide peaks were at a very low concentration and perhaps competing for charge. These data demonstrated how the rapid scan is especially advantageous for the analysis of polymer mixtures at a low level as well as for a

Figure 7. Intensity of angiotensin I, [M + 2H]2+, versus mass or rf control voltage in (a) normal scan mode and (b) rapid scan mode. A mass isolation window of ∆m/z 20 was centered at m/z 649 and used for both the normal and rapid scans. The isotope peaks for doubly charged angiotensin are observed in the normal scan trace, but not in the rapid scan trace where only the secular and beat frequencies are observed.

Figure 8. Electrospray mass spectra of 40 fmol/µL apomyoglobin in 50:50 acetonitrile/water and 0.1% acetic acid, where (a) 1 normal scan, (b) 46 normal scans collected in 74 s, (c) 1 rapid scan, and (d) the 247 rapid scans collected in 69 s. The LCQ was set to full scan MS mode with a mass range from m/z 150 to 2000. Each scan was an average of three microscans. The insets show the deconvoluted masses. In the normal scan collected over 5.6 s the molecular weight could not be determined, but in the normal scan this was possible in 3.2 s. The S/N improvement is clearly observed in the comparison of (b) with (d).

large distribution of multiply charged protein ions as discussed earlier. Data-dependent scanning is an ideal use of the rapid scan mode. In one series of scans, the first mass spectrum is a rapid scan used to select the precursor ion and the next scan is a MS/

MS scan used to collect the product spectrum. Because the rapid scan is of high sensitivity and allows for the collection of many scans in a very short time period, this scan allows for a faster selection of the precursor ion, which is required during fast chromatography of hundreds of peptides. Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

1669

Figure 9. Electrospray MS of an unknown low-amount peptide mixture. The mass spectrum in (a) shows no identifiable peptides or polymer peaks even after averaging 162 s of normal scans, and (b) shows that the rapid scan reveals a useful identification of a PEG polymer, which was collected in 36 s. The AGC target number was set to the same value of 5.0 × 106 for both scan modes.

Quantitation Using Flow Injection Analysis and the Rapid Scan. Since the rapid scan mode results in higher sensitivity and improved signal averaging, it should be a valuable scan for use in quantitative analysis. To show this quantitative advantage we collected three FIA experiments each with five injections of 5 µL of angiotensin I at 0.5 pmol/µL angiotensin I at a flow rate of 50 µL/min. The LCQ was operated in SIM mode with a center m/z of 433 and an isolation width of ∆m/z 20 to collect the signal of the triply charged ion. The three runs consisted of a normal scan and two rapid scans with manually set ion injection times of 4, 4, and 160 ms, respectively. Since the ion injection times were the same for the first two experiments, we expected them to have approximately the same total ion current (TIC) peak areas and heights as shown by the oscilloscope data presented previously. However, the data showed that the TIC peak areas and heights were 1.3 times larger in the rapid scan mode. Undersampling of the mass peaks in the rapid scan mode does not account for this discrepancy. As a result, comparison of quantitative data acquired between these two scan modes should be avoided. The rapid scan TIC peak areas and heights at an ion injection time of 160 ms are larger than the data collected at ion injection time of 4 ms by 62× and 55×, respectively, rather than the expected increase of 40×. Without an intimate knowledge of how the TIC is calculated, we were not able to elucidate the reason for these differences. Finally, the rapid scan was shown to improve the standard deviation for TIC measurements by a factor of 2.6 and the detection limit by 5 times while under the influence of chemical noise. 1670 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

CONCLUSIONS The overall signal improvement when using the rapid scan rather than a normal scan results from the product of a number of factors. We measured a 5.5× improvement in the m/z peak height, a 40× improvement in the number of trapped ions allowed before the onset of the deleterious effects of space charge, and an increased S/N ratio by n1/2, where n equals the number of scans collected over what can be collected in the normal scan range. Thus, the net ion peak signal height increase is ∼220 times for a 3D quadrupole field ion trap. Although the signal can be improved by orders of magnitude when the rapid scanning technique is used, two important limitations exist. First, the resolution is reduced by a factor of 6 so that even singly charged isotope peaks are no longer separated, and second, the mass accuracy, at least with the current data system, is reduced. The rapid scan mass accuracy limitation is at least partially due to three systematic errors: undersampling of mass peaks due to an insufficient digitization rate, an improper calibration, and a potential discrepancy in the scan functions. Correcting for the calibration masses improves the mass accuracy from 0.06 to 0.03% for apomyoglobin at 17 000 Da, and a correction of the predicted scan function error and undersampling error should improve the mass accuracy to near 0.01% for average molecular weight determinations. Despite the resolution and the current mass accuracy limitations, the rapid scan does have the advantage that it can significantly improve the signal-to-noise ratio, ion statistics, and scan time making protein molecular weight determinations at low

amounts possible. The significant S/N advantage allows the mass spectrometrist to observe ions in the spectrum that cannot be observed with confidence at low levels using the normal scan. The rapid scan ensures the operator that there is not an instrumentation problem and it can be used to easily locate a weak precursor ion signal for a MS/MS scan. The advantage for datadependent scans especially under time-limited chromatographic separations is considerable. And finally, with a linear ion trap28 one would expect to improve the ion signal for the rapid scan by another order of magnitude. One would anticipate that you could measure protein molecular weights at the low-attomole level. (28) Bier, M. E.; Syka, J. E. P., Ion Trap Mass Spectrometer System and Methodology. U.S. Patent 5,420,425, 1995

ACKNOWLEDGMENT The authors thank National Science Foundation for Grant DBI9729351 and Carnegie Mellon University for support in the purchase of the Thermo Electron LCQ. Note Added after ASAP Publication. In the paragraph discussing Figure 8, parts b and c were incorrectly cited in the version published ASAP February 11, 2005; the corrected version was published ASAP on February 15, 2005.

Received for review September 15, 2004. Accepted December 2, 2004. AC048636M

Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

1671