Gain Switching for a Detection System to Accommodate a Newly Developed MALDI-Based Quantification Method
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In matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), matrix-derived ions are routinely deflected away to avoid problems with ion detection. This, however, limits the use of a quantification method that utilizes the analyte-to-matrix ion abundance ratio. In this work, we will show that it is possible to measure this ratio by a minor instrumental modification of a simple form of MALDI-TOF. This involves detector gain switching.
KeywordsDetector gain switching MALDI-TOF instrument MALDI quantification
Recently, we reported a method to quantify an analyte (A) by utilizing matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry [1, 2]. The method is based on the observation that the analyte-to-matrix ion abundance ratio, I([A + H]+)/I([M + H]+), to be called the ion ratio in this work, is proportional to the amount or concentration (c([A])) of the analyte in an analyte-matrix mixture. Four main features and/or requirements for a reliable quantification based on the ion ratio are (1) sample homogeneity, (2) control of the effective temperature in the early matrix plume, (3) construction of the calibration curve by plotting I([A + H]+)/I([M + H]+) versus c([A]), and (4) to quantify only those samples with the degree of matrix suppression (S) smaller than a critical value. S was defined as 1 − I([M + H]+)/I0([M + H])+ with I and I0 representing the matrix ion abundances in the presence and absence of analytes, respectively. We have found that the above quantification method was applicable to mixtures and heavily contaminated samples also . S has been found to provide a good guideline to check whether a heavily contaminated sample can be quantified by the present method. One difference between the ion ratio method and other MALDI-based quantification methods is that the former method requires the abundance data not only for [A + H]+ but also for [M + H]+.
For a sample with a low analyte concentration, its MALDI spectrum is dominated by matrix-derived ions. When one attempts to measure the abundances of both analyte- and matrix-derived ions in such a case, one may encounter a problem related to the linear dynamic range of the detection system. This can occur when using commercial MALDI-TOF equipped with a detection system incorporating a single 8-bit analog-to-digital (A/D) converter. In most studies involving MALDI, however, one wants to improve the spectral data only for analyte-derived ions. Therefore, an operator routinely deflects away the matrix-derived ions and adopts a high detection gain for analyte-derived ions .
In the instruments built in this laboratory, the above problem has been handled in two ways. The first method, used with our instruments at present, involves the use of an A/D with a large dynamic range, a dual 10-bit A/D to be specific. This is costly. An alternative is to provide two different tracks and detection systems, one for the matrix-derived ions and the other for the analyte-derived ions . This dual-track instrument is even more costly. Another problem related to the linear dynamic range of a detection system is the saturation of the detector used, which is usually a microchannel plate (MCP) .
In this work, we used a detection system consisting of a single MCP and a single 8-bit A/D. Then, instead of deflecting away matrix-derived ions, we switched the MCP gain from low when matrix-derived ions arrived to high when analyte-derived ions arrived. We will show that such a minor modification allows us to construct a calibration curve displaying excellent direct proportionality between the ion ratio and c([A]).
A homebuilt MALDI-TOF instrument  was used in this work. It consists of an ion source with delayed extraction, a linear TOF analyzer, and a deflector. The ion detector was a microchannel plate (MCP, #31849; Photonis, Sturbridge, MA, USA). A 337 nm output from a pulsed nitrogen laser (MNL100; Lasertechnik Berlin, Berlin, Germany) was used as the light source. An A/D converter (DC152 Acqiris, Agilent Technologies, Santa Clara, CA, USA) was made to operate in the single 8-bit or dual 10-bit modes.
A fast, high-voltage push-pull switch (HTS 61-03-GSM; BEHLKE Electronic, Auernberg, Germany) was used to switch the MCP voltage. A schematic of the voltage switching circuit is shown in Supplementary Figure S1.
In most studies, a liquid matrix consisting of 3-aminoquinoline (3AQ) and α-cyano-4-hydroxycinnamic acid (CHCA) in methanol was used [6, 7]; 1.0 μL of the methanol solution contains 320 nmol of 3AQ and 40 nmol of CHCA, referred to here as 3AQ/CHCA. Methanol was the solvent in the analyte solution as well. Identical volumes of the matrix and the analyte solutions were mixed to prepare a sample solution; 1.0 μL of the sample solution was loaded onto the hydrophobic part of an anchor chip plate (ASTA, Suwon, Korea) and air-dried. Subsequently, a liquid solution containing 3AQ/CHCA and the analyte remains. The homogeneity of a liquid matrix is an advantage when using it as a matrix compared with a solid matrix. Another advantage is that the effective temperature and, hence, the MALDI spectrum also, remains constant over 10,000 or more laser shots .
A solid sample was also prepared. The analyte and CHCA were dissolved in 25% acetonitrile in water; 1.0 μL of the solution containing 25 nmol of CHCA was loaded and then vacuum-dried. The total ion count (TIC) control was used to acquire reproducible spectra .
Peptide Y5R was purchased from Peptron (Daejeon, Korea). 3AQ and CHCA were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Results and Discussion
Dynamic Range for A/D Conversion
MCP Gain Switching
Earlier, we suggested that we may be able to escape the above problem by switching the MCP gain, i.e., by using a low MCP gain when the matrix-derived ions arrive at the detector and switching it to a higher value when the analyte-derived ions arrive. In fact, a similar idea has been used to avoid detector saturation by ions appearing at low m/z values .
In practice, we can switch the detector gain by switching the MCP voltage. Because the quantity that we measure for the analyte quantification is the ion ratio, I([A + H]+)/I([M + H]+), one may think that calibration for the MCP gain is needed for the matrix and analyte ions. In actual studies, we kept the MCP voltage fixed at −1700 V for the detection of the matrix ion and fixed at −2300 V for the analyte ion. Then, MCP gains contribute to the ion ratio only as a constant multiplication.
In previous works, we fixed the MCP voltage at −2300 V throughout the acquisition of a MALDI spectrum. Hence, to compare the present result with previous ones, it is necessary to measure the MCP gain at −2300 V (G1) and at −1700 V (G2). Previously, we calibrated the MCP gain using the total charge of a single ion pulse. In this work, we used a simpler method based on the fact that the number of ions produced by liquid MALDI for a given sample remains nearly constant when the laser pulse energy is kept constant. That is, we estimated the G1/G2 ratio simply by taking the ratio of the matrix ion abundances measured at −2300 and −1700 V. The gain ratio, G1/G2, thus measured was 184 ± 14. We multiplied the matrix ion abundance measured at the MCP voltage of −1700 V by this ratio. This result is shown in Figure 1d. The MALDI spectrum acquired by the single 8-bit A/D with MCP gain switching is similar to the spectrum acquired by the dual 10-bit A/D without gain switching. Quantification results for Y5R carried out without and with MCP gain calibrations are shown in Supplementary Table S1. They are essentially the same.
We noted that the linear dynamic range for quantification would be limited when a MALDI-TOF instrument with a single 8-bit A/D was used without gain switching. A good way to compare the performance of different detection systems in the analyte quantification is to compare the calibration curves constructed with the data acquired by these systems.
We then converted the detection system to the single 8-bit A/D. We found that the MCP voltage of −2300 V was the optimal value, just as in the case of the dual 10-bit measurement. The calibration curve drawn with the data collected at this voltage with the single 8-bit A/D is shown in Figure 2b. As is evident, the curve is not linear. The linear dynamic range of the calibration curve is 10 or less.
Finally, we acquired the calibration curve for the same sample, this time using the single 8-bit A/D and with MCP gain switching. For simplicity in the measurement and data treatment, we held V1 (before switching) constant at −1700 V and V2 (after switching) at −2300 V. The slope of the curve in the log–log plot in Figure 2c, 1.04, supports the contention of direct proportionality between the ion ratio and c([A]). The data shown in the curve suggest a linear dynamic range of 3000, slightly larger than that for the dual 10-bit A/D. The reason why the linear dynamic range for single 8-bit switching detection appears wider than that with dual 10-bit detection will be described later. We also carried out similar experiments on the MALDI of Y5R using solid matrixes such as CHCA. In these cases as well, MCP gain switching produced excellent linear calibration curves even when a single 8-bit A/D was used. This result is shown in Supplementary Figure S2.
When the MCP voltage is increased, it happens eventually that the increase of its gain slows down, a phenomenon known as MCP saturation . In MALDI, the occurrence of saturation can be checked by deflecting away the ions with low m/z values, i.e., matrix-derived ions. As shown in Supplementary Figure S4, saturation affects the MALDI spectrum at an MCP voltage of −2400 V. We also observed the occurrence of saturation at an MCP voltage of −2300 V, as used to acquire the calibration curve shown in Figure 2a, whereas it was unobservable at −1700 V, as used during the single 8-bit switching measurement. An increase in the analyte ion signal occurring due to the reduction in the amount of detector saturation may be responsible for the wider dynamic range observed in Figure 2c compared with that in Figure 2a.
With regard to analyte quantification, whether detector saturation would noticeably distort the calibration curve must be considered. Hence, we constructed two calibration curves under identical experimental conditions except for the MCP voltage, i.e., −2300 and −2600 V—; the gains at these voltages differ by ~3. The two calibration curves acquired with the two MCP voltages are compared in Supplementary Figure S3. It is remarkable to note that the slopes of the two curves in the log–log plot, 0.974 and 1.01, respectively, are nearly the same. We do not have an explanation for the close similarity of the two curves. Further studies are therefore needed.
In the ion ratio method for analyte quantification by MALDI-TOF, the abundances of both the analyte- and matrix-derived ions are needed. These are difficult to measure simultaneously because their abundances often differ greatly. In this work, we avoided this problem by switching the gain of the MCP detector used. We expect that most instruments equipped with a single MCP and a single 8-bit A/D can accommodate the ion ratio method after minor modifications.
This work is supported by Institute for Basic Science (IBS-R006-D1).
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