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Frequency Scan of a Quadrupole Mass Analyzer in the Third Stability Region for Protein Analysis

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Feature Article

Frequency Scan of a Quadrupole Mass Analyzer in the Third Stability

Region for Protein Analysis

Z. X. Niea,b,c( ), C.-W. Lina,d( ), W.-P. Penga( ), Y. T. Leea,d( ) and H.-C. Changa* ( )

a

Institute of Atomic and Molecular Sciences, Academia Sinica, P.O. Box 23-116, Taipei 106, Taiwan, R.O.C. b

State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, P. O. Box 71010, Wuhan 430071, P. R. China

c

Department of Physics, Wuhan University, Wuhan 430072, P. R. China d

Department of Physics, National Taiwan University, Taipei 106, Taiwan, R.O.C.

A linear quadrupole mass spectrometer operating in the third stability region [(a, q)» (3, 3)] has been developed and utilized to obtain the mass spectra of multiply charged protein ions generated by electro-spray ionization. The spectra were acquired by scanning the frequency of the quadrupole with a home-built wide-band power amplifier from 400 to 100 kHz at constant voltage amplitudes in the range of 100 V. A mass resolution approaching R1/2= 200 was achieved for doubly charged gramicidin S ions at m/z 570.5. Further improvement of the resolution is anticipated when a higher-voltage power amplifier is available.

Keywords: Quadrupole mass analyzer; Frequency scan; Proteins.

INTRODUCTION

Mass spectrometry, a core technique in current pro-teomics,1has made tremendous progress in recent years since the development of soft ionization sources such as electrospray ionization (ESI)2and matrix-assisted laser desorption/ionization (MALDI).3 There are four basic types of mass analyzers used in the current proteomics re-search: linear quadrupole, time-of-flight, quadrupole ion trap, and Fourier transform ion cyclotron resonance mass spectrometers.4The linear quadrupole analyzer is most widely used because of its many attractive features: high sensitivity, moderate mass resolution and mechanical sim-plicity in a compact device. The spectrometer, when com-bined with ESI to produce multiply charged ions, holds promise to analyze very large biomolecules or even biolog-ical whole cells.5-8However, to obtain the mass spectra in high m/z (mass-to-charge ratio) regions, lowering the radio frequency (RF) of the ac field applied to the quadrupole is needed. Beuhler and Friedman9have studied the formation of high-mass water cluster ions up to m/z 59,000 with the quadrupole operating at 292 kHz. Using the same type of

instrument, Smith and coworkers10 observed multiply charged bovine cytochrome c and porcine pepsin ions in the range of 45,000.

In extension of the m/z range, operating the mass ana-lyzer in the amplitude scan (AS) mode at lower frequencies is an effective approach, while operating the analyzer in the frequency scan (FS) mode at constant voltage amplitudes is the other. The feasibility of frequency scan was first dem-onstrated by Paul et al.11,12nearly 50 years ago. The authors swept the frequency over a small range, from 2.38 to 2.54 MHz at a constant voltage amplitude (925 V), and success-fully observed the mass spectrum of85Rb and87Rb isotope ions.11,12However, the difficulty in electronics hampered the implementation of the FS mode. No work on the fre-quency scan of a linear quadrupole was reported recently except for atomic and small molecular ions.13The resolu-tion typically achieved was 150 at m/z 100. Again, the diffi-culty of obtaining higher-resolution mass spectra hindered the development of the FS quadrupole mass spectrometer, even though the method appears quite attractive for mass analysis of very large biomolecules.

Our group has engaged in development of

nanopar-Special Issue for the 4thAsia Photochemistry Conference, January 5~10, 2005, Taipei, Taiwan, R.O.C. * Corresponding author. E-mail: hcchang@po.iams.sinica.edu.tw

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ticle mass spectrometry for several years.14-19 A laser-induced fluorescence/ion trap (LIF-IT) was proposed as a detector to overcome the ineffectiveness of conventional ionization-based detectors for nanoparticle studies.18The LIF-IT, in principle, can be coupled to the quadrupole eas-ily to detect any fluorescent or fluorescently labeled nano-sized particles.19This development motivated us to explore the possibility of operating the quadrupole in the FS mode to obtain the mass spectra of high-mass biomolecules. A preliminary test with the quadrupole scanned in the first stability region yielded a mass resolution in the range of 50 for protein ions with m/z 1000. To improve the resolution, attempts were made to conduct the measurement in the higher stability region. According to Dawson,20region I is closest to the origin and is of practical interest for mass analysis (Scheme I). Almost all commercial quadrupole mass analyzers operate at the tip of this region because low-est dc voltages (U) and ac voltages (V) are required to be applied to the quadrupole. In a quadrupolar field, the mo-tion of an ion in the x and y direcmo-tions is described by the Mathieu equation,21

(1)

where

(2)

(3)

andx = Wt/2, e is the charge carried by the ion, m is the mass of the ion,W is the angular frequency of the applied RF voltage, and r0is the radius of the circle tangent to the four hyperbolic electrodes, u is the transverse displacement in the x and y directions from the center of the field, and a, q are dimensionless variables. In region I, the stability values at the tip point are a = 0.23699 and q = 0.70600. Region II is the area bordering the abscissa with a = 0 and 7.514 < q < 7.580. Region III is a non-linear quadrangle with an upper tip M at (a = 3.1643, q = 3.2341) and a lower tip S at (a = 2.5212, q = 2.8153). To obtain the mass spectrum, the oper-ating line or the mass-scan line with a slope ofl = a/2q = U/V should pass through the origin of the (a, q) diagram.

A number of experiments have demonstrated that mass resolution of a quadrupole can be greatly improved if the device is operated in the higher stability region.22-28A

resolution up to 4000 has been achieved in Region III for 59

Co+.25However, all previous results were obtained in the AS mode for atomic or small molecular ions with m/z < 100. The reason for this restriction is that if larger ions are to be analyzed, a much higher voltage must be applied. For ex-ample, if the quadrupole is to be operated in Region III, the applied voltages (both U and V) should be nearly tripled. This 3-fold (or higher) voltage increase becomes quite im-practical when analyzing protein ions with m/z in the range of 1000. In contrast to the AS mode, these regions can be easily accessed with the quadrupole operating in the FS mode.13We report in this communication that it is possible to establish such an operation by scanning the frequency of a wide-band power amplifier that drives the quadrupole at constant voltage amplitudes (Scheme II). The protein sam-ples used to demonstrate this concept are gramicidin S and cytochrome c.

EXPERIMENT

Proteins were obtained from Sigma (St. Louis, MO) and used without further purification. They were dissolved in 30% (v/v) methanol/water to a concentration of 100mM gramicidin S and 10mM cytochrome c separately for ESI. Scheme I Stability diagram for a quadrupole mass

analyzer. Regions giving ion motions si-multaneously stable in the x and y direc-tions are shaded in gray. They are indi-cated as the first (I), second (II) and third (III) stability regions, respectively

2 2 ( u 2 ucos 2 ) 0, d u a q u dx + - x = 2 2 0 8 , u x y eU a a a m r = = - = W 2 2 0 4 , u x y eV q q q m r = = - = W

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The solutions were kept below 5°C to prevent degradation before use.

The experiment was performed using a quadrupole mass spectrometer equipped with a home-built ESI source in a design similar to that described previously.29The spec-trometer consisted of a commercial quadrupole mass filter from Extrel (Merlin, 150-QC). A syringe pump (SGE, Model 362) delivered the sample solution to the ESI probe (Fisons Instruments, VG Platform) at a constant infusion rate of ~6 mL/h. The ESI probe comprised a needle (200mm i.d.) with dry nitrogen as the nebulizing gas. A ~5 kV dc voltage was applied to the needle with respect to a capillary, which was kept at the ground potential and heated to ~100°C for ion desolvation. The separation between the needle and the capillary (0.75-mm i.d. and 220-mm length) was ~5 mm. Carried by the gas flow, the ESI-generated ions passed through the heated capillary, the first skimmer (0.60-mm orifice diameter), the second skimmer (0.80-mm orifice di-ameter), and were finally focused by a set of Einzel lenses to the entrance of the quadrupole filter. The typical dc volt-ages applied to the skimmers were 50-100 V. A mechanical pump (Edwards, E1M80) evacuated the first differential re-gion to a pressure of ~1 Torr, followed by evacuation of the second stage to ~50 mTorr with a Roots blower (Alcatel, RSV150B) and finally to ~5´ 10-6Torr in the detection chamber with a turbo-molecular pump (Osaka, TG350).

Frequency scan of the quadrupole was conducted with a replacement of the RF power supply of the 150-QC quadrupole mass analyzer by a home-built power

ampli-fier.30The amplifier, driven by a synthesized function gen-erator (Stanford Research, DS345), provided RF voltages with variable frequencies at constant amplitude. Within the frequency range of 100-500 kHz, the ac peak-to-peak am-plitude was adjustable from 0 to 400 V without significant distortion of the waveforms. The dc voltage, on the other hand, was kept constant during the scan throughout the en-tire experiment (Scheme II). Prior to the mass scan, adjust-ments were made to obtain the optimal ion transmission and resolution. The ions, after passing through the mass an-alyzer, were detected by a channeltron biased at -2600 V in an ion counting mode. The typical ion intensity after opti-mization was 1,000 counts/s as a total discharge current of 1mA was applied between the ESI probe and the capillary.

RESULTS AND DISCUSSION

Fig. 1 shows the mass spectra for gramicidin S (m = 1141) in the m/z range of 500-650. The first spectrum was obtained with the mass analyzer operating in the regular AS mode at the frequency of 880 kHz (Fig. 1a). It comprises only a single peak corresponding to the doubly charged ion at m/z 570.5. The typical mass resolution achieved is R1/2= 400, with the definition

(4)

whereDm1/2andDW1/2are the peak widths at the half height for the spectra obtained in the AS and FS modes, respec-tively. The corresponding FS spectrum obtained in the first stability region with U = 16.30 V and 2V = 206.3 V (U/V = 0.1580) over the frequency scan range 400-340 kHz is shown in Fig. 1b, where a broad band appears at 371.84 kHz. The band has a half-height width of 10.56 kHz, giving R1/2= 18. The resolution can be significantly improved to R1/2= 60 by adjusting the applied voltages to U = 16.40 V and 2V = 206.1 V (U/V = 0.1591) (Fig. 1c). This improve-ment in mass resolution can be qualitatively understood from the equation11,20

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wherel = U/V. In region I, if the quadrupole operates along the line that passes through the origin of the (a, q) diagram with a constant slope (a/2q = U/V), the maximum value ofl islmax= 0.168, given the stability tip at (a, q) = (0.23699, Scheme II Frequency scan of a quadrupole mass

an-alyzer with the ac and dc voltages dis-played on the left and right axes, respec-tively. 1/ 2 1/ 2 1/ 2 , 2 m R m W = = D DW 1/ 2 1/ 2 max 0.751 , 1 / m R m l l = = D

(4)

-0.70600).

In region III, the quadrupole operates at the trap pa-rameters approximate to (a, q) = (3, 3). Compared to the first stability region,lmax= 0.168, not only the absolute values of a and q increase more than 3-fold, but also the ra-tio between ac and dc voltages along the operating line in-creases substantially. To achievel » lmax= 0.489 near the upper tip, we increased the applied dc voltage and simulta-neously decreased the applied frequency. When the fre-quency was scanned from 200 to 150 kHz at U = 46.20 V and 2V = 203.1 V (l = 0.455) in the third stability region, a sharp feature emerged for the doubly charge gramicidin S, as shown in Fig. 1d. The resolution is R1/2= 103 for the peak with a central frequency of 171.77 kHz and a half-height width ofDW1/2= 0.80 kHz. Further increase of the voltages to U = 66.20 V and 2V = 286.0 V (l = 0.462) sharpened up the feature to R1/2= 184 (Fig. 1e). In accord with the theory, operating the quadrupole analyzer in the third stability region promotes the resolution of the mass spectra. The trade-off is that the ion intensity decreases by about a factor of 10, compared to that obtained in the first stability region.25

Cytochrome c was chosen as the target protein to cali-brate the mass spectrum in the higher m/z region. Fig. 2a shows the ESI mass spectrum of horse heart cytochrome c (m = 12359 Da), obtained by scanning the voltage ampli-tude at a constant frequency (880 kHz). The spectrum dis-plays a relatively narrow charge state distribution from +7 to +11. The resolutions achieved are R1/2= 187, 166, 184, 177, and 147 at m/z 1125, 1234, 1374, 1546, and 1767, re-spectively. The corresponding FS spectrum obtained in the first stability region is given in Fig. 2b, where the peaks ap-pear atW/2p = 313.99, 299.73, 284.64, 268.61, and 251.10 kHz with U = 22.75 V and 2V = 287.5 V (l = 0.158). The resolutions of the individual peaks are R1/2= 28, 24, 69, 48 and 28, respectively. Again, the spectrum is better resolved when the frequency scan is carried out in the third stability region (Fig. 2c) with U = 41.50 V and 2V = 178.9 V (l = 0.464). Transmission of these five ions occurs at 115.43, 110.4, 104.70, 98.63, and 92.12 kHz with the resolution in-creased to R1/2= 38, 79, 68, 90 and 66, respectively.

It is instructive to relate the experimentally measured W and V to m/z of the individual ions investigated in the third stability region. According to eq. (3), V/W2

is linearly proportional to m near the tip of the stability diagram, where q is a constant. Indeed, our experimental data show a linear relationship between these two parameters for both

Fig. 1. ESI mass spectrum of gramicidin S, obtained by (a) amplitude scan of the quadrupole at the con-stant frequency of 880 kHz, (b,c) frequency scan of the quadrupole in the first stability re-gion from 400 to 340 kHz with U = 16.30 V and 2V = 206.3 V (b) and U = 16.40 V and 2V = 206.1 V (c), and (d,e) frequency scan of the quadrupole in the third stability region from 200 to 150 kHz with U = 46.20 V and 2V = 203.1 V (d) and U = 66.20 V and 2V = 286.0 V (e).

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gramicidin S and cytochrome c, i.e. V/W2

= km, where k is a constant (Fig. 3). We obtain k = 5.954± 0.009 ´ 10-6, with an uncertainty of±0.15%. It leads to the conclusion that af-ter proper calibration with model proteins, the mass mea-surement accuracy of the FS quadrupole mass spectrometer over m/z = 500-2000 is better than 0.2%.

In the third stability region, the mass resolution is mainly limited by the number (n) of the RF cycles experi-enced by the ions in the quadrupole,20,31

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wheret is the time of one RF cycle and tris the residence

time of the transmitted ion. Sincet = 1/2pW and tris related

to the rod length (L) of the mass filter and the ion accelera-tion energy (Vz) as

(7)

it is easy to show

(8)

For a given q, the equation can be rewritten to

(9)

where C is a constant. The equation predicts that R1/2is lin-early proportional to amplitude of the ac voltage but inde-pendent of the frequency applied to the quadrupole. In Fig. 3, we also show the mass resolution R1/2after scaling by V. Although the value of this parameter (R1/2/V) fluctuates no-ticeably, it stays fairly constant over the m/z = 500-2000 in-vestigated in this experiment. The result is in a reasonable agreement with the theory mentioned above and also in line with the observation in the first stability region by Rolando et al.13for atomic ions and molecular ions with m/z < 200.

To conclude, we have demonstrated that it is possible to obtain the mass spectra of protein ions by operating the linear quadrupole mass analyzer in the third stability re-gion. The highest resolution we have been able to achieve is ~200 at m/z 570 with the mass measurement accuracy ap-proaching 0.1%. With the continued development of the wide-band power amplifier, further improvement on the performance of the frequency scan quadrupole mass spec-trometer is practical.

Fig. 2. ESI mass spectra of cytochrome c, obtained by (a) amplitude scan of the quadrupole at the con-stant frequency of 880 kHz, (b) frequency scan of the quadrupole in the first stability region from 320 to 220 kHz at U = 22.70 V and 2V = 287.5 V, and (c) frequency scan of the quadru-pole in the third stability region from 120 to 85 kHz at U = 41.50 V and 2V = 178.9 V.

Fig. 3. Variations of V/W2(U) and R

1/2/V (+) with m/z

in the frequency scan of the quadrupole mass spectrometer for gramicidin S and cytochrome c.

2 2 1/ 2 r , t R n t æ ö µ = ç ÷ è ø , 2 / r z z L L t eV m u = = 2 2 1/ 2 . z m R L eV µ W × 2 1/ 2 2 0 , z L V R C r V =

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ACKNOWLEDGEMENTS

We thank Academia Sinica and the National Science Council (Grant No. NSC 92-2113-M-001-047) of Taiwan for financial support. ZXN thanks Profs. X. W. Zhu and K. L. Gao at Wuhan for helpful discussions.

Received April 26, 2005.

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數據

Fig. 1 shows the mass spectra for gramicidin S (m = 1141) in the m/z range of 500-650
Fig. 2. ESI mass spectra of cytochrome c, obtained by (a) amplitude scan of the quadrupole at the  con-stant frequency of 880 kHz, (b) frequency scan of the quadrupole in the first stability region from 320 to 220 kHz at U = 22.70 V and 2V = 287.5 V, and (

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