Anal. Chem. 1995,67,1034-1041
Improvement of Signal Reproducibility and Matrix/ Comatrix Effects in MALDI Analysis Atlcady 1. GUSBV,William R. Wilkinson, Andrew Proctor, and David M. Hercules**t Department of Chemistty, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
The main problems associated with matrix-assisted laser desorptionhonization (MALDI) quantitative analysis are poor shot-to-shot reproducibility, crystal inhomogeneity, signal degradation, and data acquisition system nonlinearity. These problems are addressed in this paper. The instrumental setup was modified to obtain required linearityand measurement accuracy over a dynamic range of lo3. To homogenize the crystal structure, ferulic acid (FA) and fucose were used to form a binary matrix. The optimum molar ratio was analyte/FNfucose = 1:5000: 5000. 2,5-Dihydroxybenzoic acid (DHB), fucose, and 5-methoxysalicylicacid (MSA) were used to form a multicomponent matrix, the optimum analyte/DHB/fucose/ MSA molar ratio was 1:104:(3 x 103-1 x 104):500. Another preparation method that worked particularly well with DHB involved accelerated drying in high-flow nitrogen at room temperature. Application of these methods gave significant enhancement in spectral reproducibility, signal intensity, and mass resolution. A relative standard deviation of 6-12% was obtained with a mass resolution of 350-400 (fwhm) at ~ 6 0 0 0Da. The FA and DHB comatrices were compared with various matrices and a standard preparation of DHB and FA. The comatrices showed a ca. 50%increase in signal intensity, a 300% decrease in relative standard deviation, and a 30-40% higher resolution. In addition, the use of comatrices resulted in less signal degradation as a function of the number of laser shots in the same location. The visual and spectral differences in crystal structure between FA and DHB both with and without comatrices are illustrated. The laser power was optimized to avoid high abundance of cluster ions and to increase molecular signal. Based on the data presented, an analytical protocol for MALDI quantitative analysis was developed. Matrix-assisted laser desorption/ionization (MALDI) time-offlight (TOF) mass spectrometry is a soft ionization method and lends itself well to the investigation of biomolecules. MALDI has become a powerful tool for the analysis of nonvolatile, high-mass The primary application of the method to date has been the determination of molecular weight, using the unique ability of the soft ionization to allow analysis of large fragile molecules without fragmentation. + Present address: Department of Chemistry, Vanderbilt University, Box 1822 Station B, Nashville, TN 37235. (1) Vertes, A; Gijbels, R In Lnser Ionization Mass Analysis; Vertes, A, Gijbels, R, Adams, F., Eds.; John Wiley & Sons: New York, 1993; p 127. (2) Hillenkamp, F.; Karas, M.; Beavis, R C.; Chait, B. T. AndChem. 1991, 63, 1193A
1034 Analytical Chemistty, Vol. 67, No. 6, March 15, 1995
Mass measurements of high molecular weight proteins and peptides mixtures,' oligosaccharides,3enzymatic protein digests? underivatized DNA oligomer^,^ and tryptic maps6 are typical applications. The absolute sensitivity of the analysis was reported to be in the femtomole range,7 and the mass range was expanded to 200 000 Da.* Thus, the application of MALDI to quantitative analysis should be extremely useful. However, such applications have been demonstrated only r e ~ e n t l y . ~ - ' ~ There are several problems associated with MALDI quantitative analysis. The signal level strongly depends on laser beam homogeneity and irradiance,I6 with a tendency for the signal to increase exponentially with increasing laser energy.I7 Sample preparation, primarily the crystallization processes and substrate surface conditions, also leads to poor shot-to-shot and sample-tosample reproducibility. Additional errors may appear due to unavoidable instrumental errors, such as detector nonlinearity and saturation effects of the matrix or analyte peaks. Saturation of the detector limits the dynamic range. Therefore, the detector voltage must be maintained such that the detector response remains linear in a low-amplification range, which limits the signalto-noise ratio for high molecular weight components. Quantitative measurements using an external standard have shown rough linear correlation in the range 1-10 pmol for oligosaccharide in 3-amino-4hydroxybenoic acid (3,4AHB);loan analogous result was reported for 5'-AGTC-3' in nicotinic acid." However, an external standard cannot compensate for the previously mentioned problems. An internal standard can be employed to avoid these problems. The internal standard method greatly (3) Stahl, B.; Steup, M.; Karas, M.; Hillenkamp, F. Anal. Chem. 1991,63,1463. (4) Sch&, M.; Btimsen, K 0.;Gassmann, E. Rapid Commun. Muss Spectrom. 1991,5,319. (5) Wu, K J.; Steding, A; Becker C. H. Rapid Commun. Mass Spectrom. 1993, 7,142. (6) Billeci, T.M.; Stults, J. T. Anal. Chem. 1993,65, 1709. (7) Stmpat, K,; Karas, M.; Hillenkamp, F. Znt. J. Mass. Spectrom. Zon Processes 1991,111, 89. (8)Karas, M.; Bahr, U.; Ingendoh, A; Hillenkamp, F. Angew. Chem., Int. Ed. Engl. 1989,10, 359. (9) Nelson, R W.; McLean, M. A.; Vestal, M. L. Proceeding ofthe 40th ASMS Conference on Mass Spectrometry and Allied Topics, Washington, DC, 1992; American Society for Mass Spectrometry: Santa Fe, NM, 1992; pp 19191920. (10) Harvey, D. J. Rapid Commun. Mass Spectrom. 1993,7, 614. (11)Tang, K; Allman, S. L.; Jones, R B.; Chen, C. H. Anal. Chem. 1993,65, 2164. (12) Preston, L. M.; Murray, K K; Russel, D. H. Biol. Mass. Spectrom. 1993, 22,544.
(13) Gusev, A. I.; Wilkinson, W. R; Proctor, A; Hercules, D. M. Appl. Spectrosc. 1993,47, 8. (14)Duncan, M. W.; Matanovic, G.; Cerpa-Poljak, A. Rapid Commun. Mass Spectrom. 1993,7, 1090. (15) Nelson, R W.; Mckan, M. A; Hutchens, T. W. AnaLChem. 1994,66,1408. (16) Ens, W.; Mao,Y.; Mayer, F.; Standing, IC Rapid Commun. Mass Spectrom. 1991,5,117. (17) Beavis, R C. Org. Mass Spectrom. 1992,27, 653. 0003-2700/95/0367-1034$9.00/0 0 1995 American Chemical Society
simplifies sample preparation and is well suited for routine analysis. Use of an internal standard enhances standard curve linearity and allows the dynamic range to be expanded to over 4 decades of concentrationll for the nicotinic acid matrix with the oligomer of Cmer (5’-AGTC-3’) as the analyte and 2-mer (5’-AG 3’) as the internal standard. Masses of the Cmer (5’-AGTC-3’) and 2-mer (5’-AG3’) are ca. 1000 amu. Application of an isotopically labeled internal standard provided a standard curve correlation coefficient of 0.95.14 However, use of such an internal standard is limited to small masses because of the low MALDI mass resolution. A measurement accuracy of 11%was reported for quantification of cyclosporin A using cyclosporin D as an internal standard with 2,5dihydroxybenzoicacid OHB) matrix.ls Application of the standard addition method and internal reference standard using sinapinic acid (SA) as the matrix showed 20% accuracy for bovine insulin over 1 decade of concentration (1-12 P M ) . ~This method has been improved, resulting in a standard deviation of ca. 15%.lSUnfortunately, this method is very laborious, since the series of samples must be prepared for each analyte, which requires a large amount of sample. Measurement of the absolute amount of an oligosaccharide with a fucosulated analogue (ca. 2000 amu) as the internal standard showed a linear correlation from 1pmol (detection limit with 3,4AHB) and from 0.1 pmol (detection limit with DHB) to 125pmol for both, with a correlation coefficient of 0.999 for several sample loadings.1° At the same time, it was shown” that application of an internal standard with chemical properties different from those of the analyte does not give sufficient accuracy and/or linearity of the standard curve. However, it was also determined that keeping the parameters of the system stable (primarily laser power density) allows the use of an internal standard with different chemical properties.I3 A 1.8%relative error of the standard curve slope was obtained for quantification of bovine insulin in the concentration range 0.75-100 pM using cytochrome c and/or insulin chain B as the internal standard.13 Thus, use of an internal standard helps to overcome some problems of quantitative analysis. However, there is still a need for new sample preparation techniques as well as instrumental protocols to allow use of this powerful method for routine quantitativeanalysis. Furthermore, MALDI quantitative analysis should be discussed with regard to the problems of (i) instrumentation, (ii) sample preparation,and (iii) instrumentalprotocol. The instrument should have the required dynamic range, accuracy, and sensitivity. Sample preparation should compensate for poor signal reproducibility and sensitivity. The instrumental parameters and analysis protocol should be optimized to provide reproducible results. The present paper reports an instrumental setup and analytical protocol for MALDI quantitative analysis concentrating on improvement of signal reproducibility by using an internal standard, and minimizing problems due to sample inhomogeneity. EXPERIMENTAL SECTION The TOF laser mass spectrometer employed in these investigations was a modified LAMMA 1000 (Leybold-Heraeus GmbH) laser microprobe using a nitrogen laser (VSL 337 ND, Laser Science Inc., Cambridge, MA) emitting at 337 nm, additional postacceleration (10 kv), and a PC-based data acquisition system. (18)Muddiman, D. C.; Gusev, A I.; Proctor, A; Hercules, D. M.; Venkataramanan, R; Diven, W. Anal. Chem. 1994, 66,2362.
/
I
Pre-amplifier
Figure I.Schematic of the experimental setup
The laser energy was measured by an energy radiometer (RT7200, Laser Precision Corp., Utica, NY). To minimize the effect of crystal inhomogeneity,the laser beam was defocused to 200 x 150 pm in all experiments. The size of the laser beam was monitored using a visual spot on a thin film of the matrix. The pressure was 1 x 10-6-2 x 106 mbar in the sample chamber and 2 x le7mbar in the analyzing chamber. A block diagram of the instrument is presented in Figure 1. The instrument contains an XYZ-manipulator and a microscope. The latter is particularly important for selecting the point of interaction of the laser with the sample. The desorbed ions were accelerated to 4 keV energy in the TOF region and space focused using an Einzel lens. To increase the sensitivity of the system, the ions were postaccelerated to 10 keV onto a discrete dynodetype secondary electron multiplier (SEM) (Thorn EMI, NJ). Quantitative measurements require a highly linear data acquisition system with a wide dynamic range. We used a W100B/ AM8238B (LeCroy Inc., Spring Valley, NY) preamp/amplifier with f.O.l% linearity over a dynamic range of more than 103. To expand the dynamic range of the system, the signal was split and differentially ampliiied before being input into the transient recorders TR1 and TR2 (200 MHz 8 x 32K channels; TR 8828 B, MM8103A, LeCroy Inc.). To increase the effective bit accuracy of the analog to digital converter (to 7 bits), the signal was passed through an analog filter with 1 kHz-20 MHz bandwidth before being input into the TR (Figurel). Calibration of the system by an external impulse generator (191, Wavetek, CA) showed a relative standard error of the standard curve slope of ca. 1%for a dynamic range of 102. This error represents accuracy of the acquisition system, the intensity measurement, and the impulse generator. Additionally, up to 200 spectra were summed to enhance the signal-to-noiseratio (S/N) and to further expand the dynamic range. Assuming that the noise is 5 relative units in one mass spectrum and the maximum signal that can be measured is 128 units (7 bits), a single shot spectrum has S/N = 25, and each channel has a dynamic range of 25 units. After 100 spectra are averaged, S/N will be increased to 250, and each channel will have a 250 units of dynamic range. Two parallel channels with different attenuation/ampliiication will have more than 1000 relative units without additional processing.l9 Analyfical Chemistfy, Vol. 67, No. 6, March 15, 1995
1035
A
Table 1 Sample Preparation and Analyte-to-Matrix Molar Ratios for Different Matrices
EtOH/ ACN/ matrix 0.1% 0.1% concn, matrix TFA TFA mg/mL
SA CA FA DHB HABA a-CHCA 3-HPA
510 510 410 1:lO 1:l 1:l 1:l
10-12 10-12 12-14 20-22 *5 %12 %25
OS7/ 0.5
molar ratio! x
mi+g rabo"
(1-1O):lO (3-5):lO (3-1O):lO (3-1O):lO (5-1O):lO (3-5):lO (1-5):lO
T
2x 5x 3x 6x e2x 6x e3x
103-2 x 103-104 103-7 x 103-2 x 103 103-1 x 10"
A
T
104 104
104 104
Analyte/matrix volume mixing ratio. b Optimum analyte/matrix molar ratio, lx.
J
SA
FA DHB
$iAHABA
CA
.-Kl 1
?! 6 0.75 7 0.5
All data acquisition and secondary processing software was written in-house (GOOGLY). Normal processing of the mass spectra includes data acquisition, mass calibration,smoothing, and peak intensity measurements. To increase the accuracy of the peak intensity measurements, a linear background was removed in measurements of peak areas. To compare the sample reproducibility (shot-to-shot and pointto-point), we calculated the relative standard deviation (RSD) of the analyteto-internal standard intensity ratios. Usually we collected 6-8 averaged mass spectra, each containing 100-200 single shot spectra. RSD for 5-10 samples was averaged. Sample Preparation. The following matrices were used in this study: 4hydroxy-3-methoxycinnamicacid (ferulic acid, FA); 3,5dimethoxy-4hydroxycinnamicacid (sinapinic acid, SA); 3,4 dhydroxycinnamic acid (caffeic acid, CA);%2,5dihydroxybenzoic acid (gentisic acid, DHB);' 3-hydroxypicolinicacid (3-HF'A);5312-(4 hydroxyphenylazo)benzoic acid (HABA);naqan&hydroxycinnamic acid (a-CHCA).23 FA, CA, DHB, and a-CHCA were purchased from Sigma Inc. (St. Louis, MO); 3-HPA, SA, and HABA were purchased from Aldrich Inc. (Milwaukee, WI). All matrices were used without further puritlcation. Matrix solutions were prepared at concentrations of 5-25 mg/mL (Table l), depending on the matrix, by dissolving in either ethanol/O.l% aqueous trifluoroacetic acid ("FA) or acetonitrile (ACN)/O. 1%TFA solution. The following proteins and peptides were used cyclosporin A (FW 1202 amu), cyclosporin D (1216 amu), bovine insulin chain B (3495.9 amu), bovine insulin (5733.5 amu), human Arg-insulin (5963.8 m u ) , horse heart cytochrome C (12 384 mu), horse heart myoglobin (18 800 mu), /?-lactoglobulin (18 400 amu) ,and bovine trypsinogen (24 000 mu). The cyclosporins were obtained from Sandoz Corp., and all other peptides/proteins were purchased from Sigma Inc. The analyte stock solutions were prepared by dissolving the sample in 1:lethanol/O.l% TFA at the highest possible concentration for application, usually 100-200 pM. Standards were prepared by serial dilutions of the stock solution. To increase sample homogeneity, the analyte and internal standard were premixed with the matrix before deposition on the spectrometer (19)Kristo, M.J.; Enke, Ch.G. Rev. Sci. Instrum. 1988,59,438. (20)Beavis, R C.; Chait, B. T. Rapid Commun. Mass Spedrom. 1989,3,436. (21)Wu,K J.; Shaler, T.A; Becker C. H. Anal. Chem. 1994,66,1637. (22)Juhasz, P.;Costello, C. E.; Biemann, K J Am. SOC.Mass Spectrom. 1993, 4, 399. (23)Beavis, R C.; Chaudhary, T.; Chait B. T.Oa.Mass Spectrom. 1992,27, 156. 1036 Analytical Chemistry, Vol. 67,No. 6, March 15, 1995
0.25 FA -
FA AD -
FA DHB AD Fucose -
DHB AD -
DHB DHB AD AD MSA FucoselMSA
Figure 2. Normalized signal intensities with various matrices for bovine insulin. Error bars show standard deviation. (A) Standard sample preparation and one-component matrices; (B) FA and DHB with/without comatrices.
probe. Typically 4-20 pL of analyte (or analyte and internal standard) was mixed with the matrix using an ultrasonicbath (60 s) and/or a vortex mixer (3 min). Aliquots of 1-2 p L of the mixture were deposited on a polished stainless steel substrate and dried in a stream of high-purity nitrogen. All matrix concentrationsand matrix-to-analytemixing ratios were optimized to achieve the best signal-to-noise ratio. Data are given in Table 1. RESULTS AND DISCUSSION
Matrix Selection and Sample Homogeneity. Initially, the signal intensities for bovine insulin chain B, bovine insulin, and cytochrome c from all matrices were compared for the case where the molar analyteto-matrix ratios and laser power had been optimized to obtain the best signal. Instead of reporting absolute laser power density, which can be different for different experimental conditions,24we will use a laser power above the ionization threshold. The threshold of ionization was determined as the minimum laser power necessary to produce a molecular ion peak with a signal-to-noise ratio of 3-4 after 20 shots. The average results of the normalized signal strengths for bovine insulin in all matrices are shown in Figure 2A. The signal was normalized to the highest intensity obtained for a multicomponent matrix, which will be discussed below. The laser power was 30% above the ionization threshold for the HABA matrix and ea. 50% above thresholdfor all other matrices. The error bars show the standard deviations for the point-to-point signal reproducibility. Relatively low signals for CA and HABA, particularly with analyte concentrations less than 1-3 pM, did not allow these matrices to be used for quantitative measurements. However, good shot-to-shot reproducibility of the signal for HABA and the good signal intensity observed for analyte concentrations> 10pM (24)Ingendoh, A; Karas, M.; Hillenkamp, F.; Giessmann, U. Int. J Mass. Spedrom. Ion Processes 1994,131,345.
indicates that the sample preparation was not truly optimized. Of
the matrices tested, the maximum signal intensity was obtained with DHB. For preliminary investigations, both positive and negative ion modes were used, but all subsequent data presented were obtained in the positive ion mode. It is well known that most of the MALDI matrices require that the operator scan the sample to find a good location with a strong signal. Generally, the crystal structure provides an indication of the signal quality (magnit~de).~ There are at least two main reasons for poor point-to-point reproducibility: (i) nonoptimum inclusion the analyte into the matrix crystal structure25 and (ii) variation of the analyte concentration and analyte/matrix ratio as a result of the crystallization process. It was found that one of the reasons for poor reproducibility involved different crystal types within the same matrix which have different analyte concentrations and analyte/matrix ratio distributions. Figure 3A presents scanning electron microscope images showing the two main FA crystal types. In visual terms, there are two types of crystals, a crystalline type (Cm and a t h i n crystalline film 0. The difference is mainly in the thickness and number of the crystals. It should be pointed out that most matrices have both ThF and C f l , along with a transition crystal type,so conclusionsfor the FA matrix follow for the other matrices also. Using all FA crystal types for relative intensity measurements increased the relative error of the standard curve slope from 1.8 to 30%13and the relative standard deviation of the analytehnternal standard intensity ratio from 10-20 to 50%. This confirmed the existence of a wide analyte/intemal standard ratio distribution in the crystals. To prove this, mass spectra were collected separately from each type of crystal for both DHB and FA. Figure 4 shows bovine insulin (51 pM, 1:3000 analyte/matrix molar ratio) mass spectra obtained with each type of FA crystal mF, Figure 4A, and CrT, Figure 4B). The DHB mass spectra of bovine insulin (51pM, 1:6OOO analyte/matrix molar ratio) in Figure 4C,D resulted from ThF and C f l , respectively. The main difference between the two crystal types is in the ratio Z(M)/Z(2M), which is much higher for CrT, where Z(nM) represents the appropriate integral intensities of the protonated and cationized molecular peaks (n = 1) and clusters (n > 1). It is known that the intensity Z(2M) and the ratio Z(M)/Z(2M) are directly proportional to the analyte concentration. Calibrating the experimental Z(M)/Z(2M) ratio to the ratio obtained with different analyte concentrations (2-51 pM analyte concentration; 1:(1.5 x lo5-3 x 103) analyte/matrix molar ratio) using one crystal type, we may estimate that the distribution of the analyte concentration in FA and DHB matrices varies by 300-600%. The experiment was carried out using the constant laser power of 50% above the threshold. The large error appears to be due to a high dependence of the Z(M)/Z(2M) ratio on system parameters and the laser power density stability (laser power stability was f 7 10%).Figure 5 shows the normalized dependence of the molecular peak intensity Z(M) and the ratio Z(M)/Z(2M) on the laser power using FA matrix and 30 pM bovine insulin concentration. The signal Z(M) and the ratio Z(M)/Z(2M) were normalized to the global maximums. The laser power is given as an increase above the ionization threshold. The worst case is shown in Figure 4E, where
Similar results were obtained with DHB matrix. It is very important to maximize the Z(M)/Z(2M) ratio to avoid possible intensity transfer from the molecular ion peak Z O to Z(2M,3M,4M). It was found that for DHB and FA matrices and analyte concentration of 5-50 pM that the optimum laser power is 40-60% above threshold when the molecular ion signal intensity Z(M) and Z(M)/ 2(M) both reach a maximum (Figure 5). In addition, it may be qualitatively useful that the analyte concentration distribution can be used to improve the signal at low analyte concentration by using ThF and, conversely, using CrT for higher analyte concentrations. The distribution of the analyte concentration leads to a change in the analyte/matrix molar ratio. The existence of such a distributionmay explain poor point-to-point and sample-to-sample reproducibility, unsuccessful attempts to use peptides with chemical properties different from those of the internal standard," and better results when only one crystal type was used.13 For quantitative measurements, this concentration distribution may really complicate the analysis. So to get reasonable accuracy, one should distinguish the type of crystal and use only one type. Sometimes this can be difficult, particularly because of the existence of the transient crystal types between CrT and ThF. Thus, for successful quantitative analysis, the problem of sample homogeneity needs to be addressed. Improving Sample Homogeneity. Several methods have already been developed to make crystalline matrices more homogeneous: premixing of the analyte with the matrix before deposition on the probe,26 additional deposition of several drops of the solvent (methanol) after complete crystallization, and scratching of the sample until the crystals start to form.1° Indeed, all of these make the sample more homogeneous and enhance shot-to-shotreproducibilitybut cannot increase sample-to-sample reproducibility. The following discussion will compare point-to-point reproducibility using the RSD of the analyte-to-internal standard ratio. Two systems were used in this study. The first was bovine insulin (1-10 pM) as analyte and bovine insulin chain B (5 pM), cytochrome e (15 pM),13 and Arg-insulin (10 pM) and as internal standards. The second system was cyclosporin A (1 pM) as analyte and cyclosporin D (3 pM) as the internal standard.@ The results of the point-to-point RSD present the average RSD range obtained for all these systems. We have tried many methods to create one type of the crystal and make it more homogeneous and found two of note. The first is based on the application of accelerated drying (AD) using a stream of high-purity nitrogen (instead of the original, more gentle flow). The nitrogen stream moves and mixes the aliquot during the crystallization process. The best result was obtained with DHB in which crystals prepared with AD became more closely packed and smaller (see Figure 3E) than with the standard sample preparation shown in Figure 3D. This method showed better signal reproducibility with DHB (see Table 2) and gave better signal intensity, Figure 2B. For FA, it helps to somewhat avoid problems associated with several types of crystals (see Figure 3A) and create one main crystal type without ThF, Figure 3B. Unfortunately,the method works well only for matrices dissolved in low-viscosity solvents.
(25) Beavis, R C.; Bridston, J. N. J. Pkys. D. 1993,26, 442.
(26) Beavis, R C.; Chait, B. T. Atial.Chem. 1990,62, 1836. Analyfical Chemistry, Vol. 67, No. 6,March 15, 1995
1037
r
I
Figure 3. Scanning electron microscope images demonstrating the crystal types for various matrices: FA (A, top left), FA with AD sample preparation (B, middle left), FNfucose with AD (C, bottom left), DHB (D, top right), DHB with AD (E, middle right), and DHB/MSNfucose (F, bottom right).
Another method to improve sample homogeneity is the addition of several matrix components to form a multicomponent matrix. In a recent investigationof protein inclusions in a matrix,25 it was proposed that the protein binds to the matrix crystal because of its amphiphilic nature with respect to the relatively hydrophobic specific crystal face to which it binds. To increase the hydrophobic protein/matrix interactions and optimize protein 1038 Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
inclusion in the matrix crystals, we used carbohydrate comatrices such as glucose,fructose, sucrose, and fucose. These comatrices have already been used for resolution enhancement with DHB.6 It is worth noting that, at the same time, the ribose and sucrose did not work with 3-HPA21 In our case, a-L(-)-fucose was preferred because of its more consistent and reproduciblecrystal structure. We obtained the best results with addition of fucose
Rel. units .-3
Table 2. Reproducibility and Rerolutlon Obtained with Different Standard Matrices and Multicomponent Matrices
v)
2
Rei.
-8000
12000
REL. UNITS
100 a,
F
-
-1
500
8000
M
150 -
=x
16000 D ~ ,
12000
16000 De.
E -
matrix SA
FA (std) FA FA DHB (std) DHB
DHB+MSAa DHB+MSAa a-CHCA SHPA HABA
2M
I
3M
i
_j
d
i
--
4M
AD
+ + + + + +zd
+zd fd
matrix concn, mg/mL 12 12 12 12
20 20 20 20 12 25 5
fucose concn, mg/mL
6-9
10-20
RSD
15-20 10-506 10-20 8-11 11-25' < 13 8-11
6-12 x16 12-18 10-14
resolution fwhmC
=330 x220 e250 300-380 250-300 =300 300-400 300-400 ~340 e360 380-400
5Methoxysalicylicacid concentration,1mg/mL. * Using all types of crystals. For bovine insulin. For both gentle drying and AD.
A---
Flgure 5. Normalized signal intensity l(nn) and /(M)//(2M) ratio on the laser power (in percent) above the threshold using FA matrix.
to FA rather than to DHB, but for both the reproducibility and resolution were improved, as can be seen in Table 2. Additionally, the signal strength was improved, Figure 2B. The FA crystal structure with the fucose comatrix is shown in Figure 3C. The crystals are similar to those of pure FA with AD (see Figure 3B), but the structure is more regular. Improvements for bovine insulin and cyclosporin systems were similar. Thus, Table 2 presents the average results of RSD of both systems. Another comatrix which was used with DHB was Smethoxysalicylic acid (MSA). MSA has been applied to improve DHB as a matrix for complex analyte mixtures.27 In our case, MSA and fucose addition to DHB helped to improve reproducibility and also improved resolution slightly. DHB with fucose and MSA crystals after AD are shown in Figure 3F. The DHB/MSA/fucose crystals
with AD are similar to those of pure DHB with AD but much more homogeneous, and therefore better reproducibility was observed, as seen in Table 2. The optimum analyte/FA/fucose molar ratio was 1:5000:5000, and the analyte/DHB/fucose/MSA ratio was very similar, 1:104:104:500. It should be pointed out that the signal intensity rapidly degrades when the analyte/matrix molar ratio decreases below the optimum. Thus the main difference in the morphological structure of DHB and FA crystals occurs after application of the AD. The fucose comatrix with FA and MSA/fucose comatrices with DHB do not change the morphology but significantly improve crystal homogeneity. Another very important result is that, with m o a e d FA and DHB, there is no problem in finding a crystal which works well. Although we did not measure sample-to-sample reproducibility,preliminary results indicated that the main error was caused by point-to-point rather than sample-to-sample deviation. A possible explanation for the improved reproducibility is that binary and multicomponent matrices produce more homogeneous crystal structures, which minimizes variation of the analytetomatrix molar ratio and analyte concentration. Another reason may be connected with the ability of fucose to coolz8and/or its ability to strengthen the hydrophobic proteidmatrix interaction. Improved resolution cannot be explained by elimination of cationized molecular ions and photoadducts. We found that comatrices improved the physical resolution of all peaks (less ion peak width), although suppression of the cationized peaks and less metastable fragments were also observed. This result is at least correct in the 1000-6000 Da mass range, where protonated and cationized peaks are easy to resolve, but we believe that this may also be valid for the higher mass range. Also, note that these results were obtained using a reflectron instrument and can be different for h e a r TOF. An accurate explanation of comatrix effects requires investigation of molecular structure similar to the experiments that have been carried out for Skz5Thus, application of the AD and comatricesdecreases the RSD for DHB and FA from 20%to better than lo%,improves signal strength, and achieves mass resolutions of 300-400 (for insulin). (27) Karas, M.; Nordhoff, E.; Stahl, B.; Strupat, IC;Hdenkamp, F. Proceeding of the 40th ASMS Conference on Mass Spectromety and Allied Topics, Washington, DC, May, 1992; American Society for Mass Spectrometry: Santa Fe, NM, 1992; pp 368-369. (28) Kiister, C.; Castoro, J. A; Willcins, C. LJ.Am. Chem. SOC.1992,114,7572.
Analytical Chemistry, Vol. 67,No. 6,March 15, 1995
1039
Rel. I
units
0.5
0
I
20
40
,
60
80
100
Number of shots
Figure 6. Shot-to-shot reproducibility (signal degradation) of the dirferent matrices. Normalized dependence of the bovine insulin signal intensity on the number of the shots in one place. A,SA; +, a-CHCA; 0,HABA; 0 , FA: 0, FA and fucose: +, DHB: 0, DHB, MSA, and fucose. Table 3. Molecular Signal (%) for Various Matrices after 100 Shots Normalized to the Maximum Intensity
matrix FA HABA SA
a-CHCA
ref 22 70 20 30
ref 29
this work
25
30 45 10 25
16
Time-Dependent Signal Degradation. Another source of possible errors in quantitative measurements is signal degradation as function of the number of shots at the same point. The average results of signal (bovine insulin) degradation with the above mentioned matrices, with and without comatrices, are presented in Figure 6. The analyte/matrix molar ratios were chosen close to the optimum of Table 1. The laser power was set close to the maximum signal strength (30%above the threshold for HABA and 40-60% above threshold for the rest of the matrices, see Figure 5). The best results were obtained with modified DHB and FA. Signal intensity with these matrices was quite stable (x20-30%) over 0-60 shots. Previously reported r e s ~ l t ofs signal ~ ~ ~ deg~~ radation using SA, FA, a-CHCA, and HABA are very similar to those reported here, Table 3. RSD of analyte (insulin)-to-internal standard (Arg-insulin) ratio was consistent from the 10th to the 80th shot. The higher RSD in several first shots can be explained by surface contamination. An increase of RSD after 80 shots was the result of the rapid analyte and internal standard signal intensity degradation. Protocol for Quantitative Analysis. Two matrices, DHB/ fucose/MSA and FA/fucose, were found to produce the best signal reproducibility: RSD and temporal signal stability. A p plication of these matrices with accelerated drying obtained RSD = 6-11%. The laser power should be kept constant in the 40-60% range above threshold. This produced the maximum molecular peak signal intensity and minimum influence of the dimer molecule on the results of analysis. However, measurement of (29) Westman, A; Demirev, P.; Huth-Fehre, T.;Bielawski, J.; Sundqvist, B. U. R Int. J. Mass. Spectrom. Ion Processes 1994,130, 107.
1040 Analytical Chemistty, Vol. 67, No. 6, March 15, 1995
the laser power density could be a problem for an instrument without sample observation. In this case, routine monitoring of the laser power would be an essential part of the analysis procedure. However, we consider sample observation a critical part of the analysis which allows precise control of the point of interaction. The selection of the crystal is not crucial for multicomponent matrices with accelerated drying. The analyteto-matrix molar ratios should be lower than 1:lO 000. To avoid possible different degradation of analyte and internal standard signals, the spectra should be collected in the linear range of the graph, as shown in Figure 6. The linear range depends on the specific matrix used but generally is between the 20th and the 50-70th shot. Skipping of the first 20 shots helps to avoid problems concerning surface contamination. Thus, based on the data presented, the following instrumental protocol was developed for quantitative analysis: 1. Determine the ionization threshold and increase the laser power 40-60% above the threshold. 2. Visually control the place of interaction. 3. Fire 20 blank shots without averaging while selecting the required attenuation/ amplification for each TR channel. 4. Accumulate the next 30-45 mass spectra in sequence with on-line sequence averaging/mass calibration (for the best resolution and accuracy possible, calibrate each spectrum and each TR channel). 5. Move the laser spot to the another crystal using the microprobe facility and visually control the new point of the interaction in the first several shots. 6. Repeat steps 3-5 until 100-200 (depending on the required S/N ratio) mass spectra are averaged. 7. Perform off-lineprocessing, which will be discussed further in another publi~ation.~~ The measurement precision ( p ) can be evaluated at the 95% confidence level using the following formula: P = f(tRSD)/W2, where t is the t statistic at a confidence level of 95%and N is the number of measurements. Assuming an ideal linear instrument response with respect to concentration and 10 measurements, the precision will be within 10%. Unfortunately, the RSD has the tendency to increase in the low and high analyte concentration ranges. In this case, the estimated precision decreased to 15%. Application of this protocol, selection of the internal standard, and examples of quantitative analysis will be published elsewhere.30 CONCLUSIONS
Several new approaches to improve MALDI space (point-topoint) and time (signal degradation) reproducibility have been investigated using a modified instrument with an expanded dynamic range. One cause of poor reproducibility is related to several different crystal types and the local differences in analyte concentration and analyte/matrix ratio. To homogenize crystal structure, we used ferulic acid (FA)/ fucose and 5methoxysalicylic acid (MSA)/fucose/gentisic acid @HB) matrices. Another method that worked particularly well with DHB was accelerated drying of the sample. Application of comatrices (fucose, MSA) with accelerated drying of the sample gave significant enhancement in reproducibility, signal intensity, and resolution. Thus the relative standard (30)Gusev, A I.; Wilkinson, W. R; Proctor, A; Hercules, D. M. Anal.Chem., submitted.
deviation of the analyte/intemal standard ratio of 6-1% was obtained with a resolution of 350-400 for several loadings. Comparison of the binary FA and multicomponent DHB matrices with various matrices such as sinapinic acid, a-cyano-4-hydrow cinnamic acid, 3-hydroxypicolinic acid ,2-(4hydroxyphenylazo)benzoic acid, and a standard preparation of DHB and FA shows improvement of at least 40-50% in signal intensity, 300%decrease in relative standard deviation, and 30-40% higher resolution. Also, measurement of signal degradation as a function of the number of shots shows the advantages of the modified DHB and FA matrices. Based on the data presented, a protocol for MALDI quantitative analysis was developed.
ACKNOWLEDGMENT This work was supported by the National Science Foundation under Grant CHE9022135. We would like to thank A G. Sharkey and D. C. Muddiman for valuable discussions and F. Hillenkamp and M. Karas for their help with instrumental (LAMMA-1000) mod~cation. The authors would also like to acknowledge the reviewers for valuable comments. Received for review May 9, 1994. Accepted January 4,
1995. AC940458H Abstract published in Advance ACS Abstracts, February 1, 1995
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