Mass and Resolution Calibration for New Triple-Stage Quadrupole Mass Spectrometers. Rob Grothe Thermo Fisher Scientific, San Jose, CA

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1 Mass and Resolution Calibration for New Triple-Stage Quadrupole Mass Spectrometers Rob Grothe Thermo Fisher Scientific, San Jose, CA

2 Overview Purpose: Robust and accurate calibration of mass positions and peak widths for a new generation of triple-stage quadrupole mass spectrometers. Methods: 1) Coarse calibration of the slope of DC/RF scan line to eliminate instrument variability; 2) Resolution calibration of DC settings to select peak width; 3) Mass calibration to place known calibrant at correct mass position. Results: Calibration of mass and resolution is demonstrated on a prototype triplestage quadrupole mass spectrometer. in positive-ion mode. Introduction Accurate analysis of a sample by quadrupole mass spectrometry requires accurate calibration of both mass and resolution. Mass calibration allows the user to correctly identify analytes in the sample (qual) and reproducibly measure their abundances (quan). Resolution calibration allows the user to select optimal peak widths for a given analysis either widening the peak width to improve sensitivity, or narrowing the peak width to improve specificity. Methods Sample Preparation Calibration mix is infused into the mass spectrometer during calibration. The calibration mix contains three polytyrosine peptides that generate ions used for calibration in both positive and negative ion modes. Mass Spectrometry The same calibration routine is used for Thermo Scientific TSQ Endura and Thermo Scientific TSQ Quantiva triple-stage quadrupole mass spectrometers. Results shown here were obtained on a prototype TSQ Quantiva triple-stage quadrupole mass spectrometer. Data Analysis Spectra are acquired while adaptively modifying the RF and DC settings. Using proprietary, purpose-built software, the spectra are fit to a deformable model of each calibrant s isotope envelope. By virtue of the model fit, accurate estimates of the ion intensity, mass position, and peak width measured at full-width half maximum (FWHM) are obtained. Semi-empirical parameters describe how mass position and peak width change in response to changes in applied RF and DC voltages. These parameters are used to adaptively adjust RF and DC to correct differences between the observed mass position and theoretical calibrant mass and between the observed and requested peak width. Calibrated settings are verified by five replicate scans measuring the RMS error in mass position and peak width against the prescribed tolerances: TSQ Quantiva triple-stage quadrupole mass spectrometer: +/- 25 mda Method Overview As shown in Figure 1, m mass analysis devices: devices are calibrated f Da) and two scan rates scan rates are calculate The process has three s calibration. The outcom of the scan line position Mathieu stability diagram FIGURE 1. Mass posit a = k DC / (m/z) M apex ( 1: Initial scan 2: Coarse cal 3: Resolution 4: Mass calib Estimation of Mass Po As shown in Figure 2, c of mass position and pe width respectively. Ther estimating the mass po A deformable isotope en The isotope envelope h horizontal scale (peak w provides estimates of m FIGURE 2. Compariso position and peak wid 2 Mass and Resolution Calibration for New Triple-Stage Quadrupole Mass Spectrometers

3 s and peak widths for a new line to eliminate instrument peak width; 3) Mass n. d on a prototype triple-. metry requires accurate lows the user to correctly sure their abundances mal peak widths for a given ity, or narrowing the peak Method Overview As shown in Figure 1, mass position and peak width are determined by setting three mass analysis devices: DET_GAIN_DAC, RES_DAC, and MASS_DAC. These three devices are calibrated for each quad (Q1 and Q3) at two peak widths (0.4 Da and 0.7 Da) and two scan rates (250 Da/s and 1000 Da/s). Settings for other peak widths and scan rates are calculated by extrapolation. The process has three steps: coarse calibration, resolution calibration, and mass calibration. The outcome of each step can be understood in terms of the manipulations of the scan line position relative to the apex of the principal stability region of the Mathieu stability diagram. FIGURE 1. Mass position and peak width determination. Mass and Resolution Calibration apex of principal stability region (q*,a*) ~ (0.706, 0.237) 2 3 Results Coarse Calibration Figure 3 shows two iterations of the DET_GAIN_DAC device, wh trend of peak width vs. m/z is as When RF/DC = 2q*/a* ~ 6, the s diagram joining the origin to the observed as a flat trend line in th When the RF/DC ratio is too low DET_GAIN_DAC is reduced by m/z to zero. The first iteration (1 a slightly negatively sloping tren desired result. FIGURE 3. Coarse calibration. g calibration. The calibration used for calibration in both a = k DC / (m/z) 4 1 q = k RF / (m/z) TSQ Endura and le mass spectrometers. antiva triple-stage d DC settings. Using eformable model of each rate estimates of the ion idth half maximum (FWHM) d peak width change in e parameters are used to n the observed mass served and requested peak suring the RMS error in nces: +/- 25 mda 1: Initial scan line (DC/RF slope too low) 2: Coarse calibration: adjust DET_GAIN_DAC to correct slope 3: Resolution calibration: adjust RES_DAC to select peak width 4: Mass calibration: adjust MASS_DAC to correct mass position Estimation of Mass Position and Peak Width As shown in Figure 2, calibration and verification involve comparing observed values of mass position and peak width to a theoretical calibrant mass and requested peak width respectively. Therefore, accurate calibration requires an accurate method for estimating the mass position and peak width for a known calibrant. A deformable isotope envelope is fit to the observed spectrum for a known calibrant. The isotope envelope has three degrees of freedom: vertical scale (intensity), horizontal scale (peak width), and horizontal shift (mass position). The model of best fit provides estimates of model parameters. FIGURE 2. Comparison of observed and theoretical values for mass position and peak width. Resolution Calibration As shown in Figure 4, resolution selected peak width. The RES_D moving the scan line closer to th peak width. The trend line of peak width vers (approximately) known in advan RES_DAC to the target peak wid linear regression. FIGURE 4. Resolution calibrat Thermo Scientific Poster Note PN ASMS13_Th095_RGrothe_e 08/13S 3

4 are determined by setting three, and MASS_DAC. These three two peak widths (0.4 Da and 0.7 ettings for other peak widths and lution calibration, and mass tood in terms of the manipulations incipal stability region of the nation. n Calibration Results Coarse Calibration Figure 3 shows two iterations of coarse calibration. Each iteration adjusts the value of the DET_GAIN_DAC device, which moves in opposition to the RF/DC ratio, until the trend of peak width vs. m/z is as flat as possible. When RF/DC = 2q*/a* ~ 6, the scan line is parallel to a line in the Mathieu stability diagram joining the origin to the apex of the principal stability region. This condition is observed as a flat trend line in the plot below (1314, yellow). When the RF/DC ratio is too low (1330, red), peak width increases with m/z. The DET_GAIN_DAC is reduced by 17 units attempting to reduce the slope peak width / m/z to zero. The first iteration (1313, blue) produces slight overcorrection, observed as a slightly negatively sloping trend line. The final iteration (1314, yellow) achieves the desired result. FIGURE 3. Coarse calibration. Mass Calibration Mass calibration adjusts the correct mass position in the DC, moving an ion along th Mass calibration is inherent RES_DAC shifts the mass mass position for the calibra ratio observed mass / theor Because coarse calibration RES_DAC value remains c avoid the need to iteratively Verification As shown in Figure 5, with MASS_DAC, RES_DAC), th estimated from five replicat counting statistics and RF j compared against the tolera FIGURE 5. Verification of = k RF / (m/z) DAC to correct slope to select peak width correct mass position lve comparing observed values rant mass and requested peak quires an accurate method for own calibrant. spectrum for a known calibrant. vertical scale (intensity), ass position). The model of best fit Resolution Calibration As shown in Figure 4, resolution calibration adjusts the RES_DAC device to achieve a selected peak width. The RES_DAC adds a voltage offset to the differential DC, moving the scan line closer to the apex of the stability diagram and thus reducing the peak width. The trend line of peak width versus RES_DAC is linear. The slope of the line is (approximately) known in advance. Five scans are taken to iteratively adjust the RES_DAC to the target peak width (e.g. 0.7) and the chosen value is selected by linear regression. FIGURE 4. Resolution calibration. al values for mass 4 Mass and Resolution Calibration for New Triple-Stage Quadrupole Mass Spectrometers

5 ation adjusts the value of e RF/DC ratio, until the the Mathieu stability region. This condition is eases with m/z. The e the slope peak width / ercorrection, observed as 4, yellow) achieves the Mass Calibration Mass calibration adjusts the MASS_DAC device to place a known calibrant at the correct mass position in the spectrum. The MASS DAC device scales both the RF and DC, moving an ion along the scan line. Mass calibration is inherently coupled to resolution calibration because increasing the RES_DAC shifts the mass position to the right. Using linear regression, we find the mass position for the calibrated RES_DAC setting and then the MASS_DAC by the ratio observed mass / theoretical mass. Because coarse calibration has established mass-independent RES_DAC tuning, the RES_DAC value remains correct even after the MASS_DAC adjustment. Thus, we avoid the need to iteratively alternate MASS_DAC and RES_DAC adjustments. Verification As shown in Figure 5, with all mass analysis devices fixed (DET_GAIN_DAC, MASS_DAC, RES_DAC), the position and peak width for each known calibrant are estimated from five replicate scans. Minimal scan-to-scan variation is observed due to counting statistics and RF jitter. The total RMS error (systematic + random) is compared against the tolerance (exterior of blue box). FIGURE 5. Verification of mass calibration. Calibration Robustness A standard reference scan line is p principal stability region as a marke differential DC is set to zero, the th adjusted by the RES_DAC. Empiri of 0.7 +/- 0.1 V sets the differential Reducing the RES_DAC from this in peak width, about 2.6 Da/V. As shown in Figure 6, a peak width 0.75 V (0.4 Da) / (2.6 Da/V) = 0.6 by setting the RES_DAC to 0.75 V FIGURE 6. Setting peak width. _DAC device to achieve a the differential DC, m and thus reducing the slope of the line is teratively adjust the value is selected by Fine calibration requires only sma makes it easy to bring new system defects. Conclusion Mass and resolution calibration of quadrupole mass spectrometers h coarse calibration routine that pro subsequent fine calibration is trivia The apex of the principal sta mass and resolution calibra Nearly all of the instrument- DET_GAIN_DAC adjustmen through the apex of the prin The standard scan line has independent, allowing easy need for iterative rounds of calibration.. Calibrated RES_DAC value determined a priori, resultin mechanical defects. All trademarks are the property of Thermo F This information is not intended to encourag intellectual property rights of others. Thermo Scientific Poster Note PN ASMS13_Th095_RGrothe_e 08/13S 5

6 ce a known calibrant at the device scales both the RF and bration because increasing the inear regression, we find the then the MASS_DAC by the pendent RES_DAC tuning, the _DAC adjustment. Thus, we RES_DAC adjustments. Calibration Robustness A standard reference scan line is produced by coarse calibration, using the apex of the principal stability region as a marker. Relative to this reference scan line, when the differential DC is set to zero, the theoretical peak width is zero. Differential DC is adjusted by the RES_DAC. Empirically, across many instruments, a RES_DAC setting of 0.7 +/- 0.1 V sets the differential DC to zero. Reducing the RES_DAC from this zero-point value produces a proportionate increase in peak width, about 2.6 Da/V. As shown in Figure 6, a peak width of 0.4 Da is achieved by setting the RES_DAC to 0.75 V (0.4 Da) / (2.6 Da/V) = 0.60V (blue line). A peak width of 0.7 Da is achieved by setting the RES_DAC to 0.75 V (2.6Da/V)*(0.7 Da) = 0.48V (red line). FIGURE 6. Setting peak width. xed (DET_GAIN_DAC, for each known calibrant are an variation is observed due to ystematic + random) is Fine calibration requires only small adjustments from calculated set points. This makes it easy to bring new systems into calibration and easily identifies mechanical defects. Conclusion Mass and resolution calibration of TSQ Endura and TSQ Quantiva triple-stage quadrupole mass spectrometers has been made simple and robust by means of a coarse calibration routine that provides a common starting point from which subsequent fine calibration is trivial and highly reproducible. The apex of the principal stability region provides a useful reference point for mass and resolution calibration. Nearly all of the instrument-to-instrument variation can be tuned out by DET_GAIN_DAC adjustment that delivers the standard scan line that passes through the apex of the principal stability region. The standard scan line has the desirable property that RES_DAC tuning is massindependent, allowing easy identification of the scan line and eliminating the need for iterative rounds of that alternate mass calibration and resolution calibration.. Calibrated RES_DAC values closely correspond to theoretical values that can be determined a priori, resulting in easy calibration and clear identification of mechanical defects. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 6 Mass and Resolution Calibration for New Triple-Stage Quadrupole Mass Spectrometers

7 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other trademarks are the property of Thermo Fisher Scientific, Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Thermo Fisher Scientific, San Jose, CA USA is ISO 9001:2008 Certified. Africa-Other Australia Austria Belgium Canada China Denmark Europe-Other Finland/Norway/Sweden France Germany India Italy Japan Latin America Middle East Netherlands New Zealand Russia/CIS South Africa Spain Switzerland UK USA ASMS13_Th095_RGrothe_E 08/13S

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