Anal. Chem. 1993, 65,2164-2166
2184
CORRESPONDENCE
Quantitative Analysis of Biopolymers by Matrix-Assisted Laser Desorption K. Tang, S. L. Allman, R. B. Jones, and C. H. Chen' Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6378
INTRODUCTION During the past few years, major efforts have been made to use mass spectrometry to measure biopolymers because of the great potential benefit to biological and medical research.' Electrospray and matrix-assisted laser desorption were developed to overcome most of the difficulties resulting from molecularfragmentation during the vaporization or ionization process. Hillenkamp and his co-workers were the first to report that large polypeptide molecules can be ionized and detected without significant fragmentation when a greater number of nicotinic acid molecules were used as a matrix.2 Since then, various groups have reported measurements of protein and biologically significant oligomers by the use of various matrices and lasers. Observation of protein with molecular masses greater than 100 OOO Dawithout appreciable fragmentation has been reported. Oligonucleotides with molecular masses greater 10 OOO Da have been successfully detected by time-of-flight mass spectrometry.3 Beavis and Chait416 have discussed characteristics of matrix-assisted UV desorption of proteins which include velocity distribution, laser power density, and chemical properties of matrix compounds. Although the theoretical details of laser desorption and ionization mechanisms of MALDI are not yet fully understood, several models have been presented to explain the production of large biopolymer ions.61' Up to now, most of the efforts of MALDI have been on detecting large biopolymers with accurate determination of molecular weight. Little progress has been made toward achieving reliable quantitative measurements of analyte, primarily because of poor shot to shot as well as sample to sample signal reproducibility. Poorly controlled sample preparation and an ion production process which is strongly dependent on laser fluence may be the major obstacles to obtaining quantitative results. It is known that the MALDI process is very sensitive to laser fluence. When laser fluence is below the desorptionlionization threshold, no signals can be observed. As laser fluence is increased to just above signal production threshold, the signal amplitude of the analyte increases almost exponentially.8 Thus, a small variation in laser fluence can cause very large fluctuations in analyte signals. When samples are prepared for a MALDI experiment, (1) Burlingame, A. L.; Maltby, D.; Russell, D. H.; Holland, P. T. Anal. Chem. 1988,60,294R2. (2) Karae, M.; Back", D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Zon Processes 1987, 78, 53. (3) Tang,K.;Allman, S. L.; Chen, C. H. Rapid Commun. Mass Spectrom. 1992, 6, 365. (4) Beavis, R. C.; Chait, B. T. Rapid Commun. Mass Spectrom. 1989, 3, 233. (5) Beavia, R. C.; Chait, B. T. Chem. Phys. Lett. 1991,181, 479. (6) Vertea, A.; Levine, R. D. Chem. Phys. Lett. 1990, 171, 284. (7) Overberg,A.;Haesenburger,A.;Hillenkamp, F.MassSpectrometry in Biological Sciences; Gross, M.; Sindona, G., Eds.; Klumber Academic Publisher: in press. (8)Beavis, R. C. Org. Mass Spectrom. 1992, 27, 653. 0003-2700/93/0365-2164$04.00/0
typically the dried distribution of matrix material is unsually not very homogeneous. For example, nonhomogeneous needle-shaped crystals were observed when 2,5-dihydroxybenzoic acid was used as a matrix.9 By observing a tremendous variation in signals produced as a result of moving the laser beam around on the surface of the sample, it became obvious that the signal level of analytes can be a strong function of the size of the spot desorbed by the laser and the location on the sample. It is also known that impurities in a sample probed by the MALDI process can cause a significant reduction in the signal elve of analytes. For example, Hillenkamp and his co-workers10 found that using ammonia acetate on a glass bead can reduce the sodium ions in a sample, thereby producing mass spectra with better resolution. In addition, the surface condition of the substrate upon which the sample is evaporated can vary the threshold for the production of plasma. In brief, it is very difficult to obtain reliable measurements of the absolute quantity of analytes by MALDI. If MALDI is going to become a routine analytical tool, it is obvious that quantitative measurement capability must be pursued.
EXPERIMENTAL SECTION A Spectra Physics (MountainView, CA) Model DCR-2A NdYAG laser capable of delivering 532-, 355-, and 266-nm light pulses was used for laser ablation. The maximum availablelaser energiesper pulse at 532,355, and 266 nm were 400,200, and 100 mJ, respectively,with correspondingpulse durations of 7,5, and 5 ns, respectively. However, typical laser fluence used in this work was less than 200 mJ/cmZto prevent any possible production of plasma. A small aperture was placed in front of the laser beam output to limit the size of the beam striking the sample target to -0.5 mm in diameter. The laser pulse energy was measuredjust before the beam went into the mass spectrometer. The laser fluencewas calculatedwith the attenuation of all optics taken into account. A linear time-of-flight (TOF) mass spectrometer was used to resolve molecular weights and detect parent analyte ions (seeFigure 1).A conversion box was used to receive ions and emit secondary electrons. A Johnston Laboratory (Cockeysville, MD) Model MM-1 electron multiplier was used to detect secondary electrons from a piece of aluminum foil in the conversion box. Signals from the Johnston multiplier went through a Ortec (Oak Ridge, TN) Model 9305 preamplifier and subsequently to a Yokagawa (Newnan, GA) Model DL2141B fast digital oscilloscope with a maximum sampling rate of 250 million samples/s. TOF wave forms were transferred to a computerfor data storage and retrieval. The meximum resolution (MIAM) of the TOF spectrometer is only -200. However, the effective resolution for these biopolymer samples is somewhat lower probably due to the formation of adducts. (9) Stupat, K.;Karae, M.; Hillenkamp, F. Znt. J. Mass Spectrom. Zon Processes 1991,111, 89. (10) Nordhoff, E.; Ingendoh, A.; Cramer, R.; Overberg, A.; Stahl, B.; Karas, M.; Hillenkamp, F.;Crain,P. F. Rapid Commun.Mass Spectrom. 1993, 6, 771. 0 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, A W S T 1, 1993 2166 DESORPTION
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Flguro 3. Negative ion spectrum of mixtures of small oligomers. The chemical structures of 2, 3, 4, and 6 mer are 5'-AW', 5'-AGT-3', I'-AQTTw', and 5'-AQTCCT-3', respectively. Nicotinic acid was used as matrix. The sample contains 120 nmoi of nicotinic acid and 30 pmoi of each oligomer. The laser wavelength was 266 nm with laser fiuence of 30 mJ/cm2. Spectrum is an average of 16 laser p u b . 160 140 -
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Moss [Doltons) Flgwo 2. Negative ion spectrum of mixtures of protein. Nicotinic acid was used as matrix. The quantity of cytochrome c (C),lysozyme (L), and myoglobin (M) used was 30 pmoi each; the quantity of nicotinic acid was 0.3 pmoi. The laser wavelength was 266 nm with fiuence of 75 mJ/cm2. Bothdimer ions and doubly charged ions were observed. Spectrum is an average of 16 laser pulses.
Oligonucleotides and protein samples used in this work were purchased from commercial companies. Oligonucleotides samples were obtained from Oligos Etc, Inc. (Wilsonville, OR). Protein sampleswere brought from Sigma, Inc. (St. Louis, MO). No further purification of commercial products was pursued. The sampleswere prepared by mixing an aqueousanalyte solution with an aqueousmatrix solution for selectedmole ratioe of matrix to analyte. The sample was put onto a stainless steel disk with a spot size of -6 mm. The data were taken within 15 min after the sample was introduced into the chamber and a vacuum of 1 X lo-' Torr was reached. Most samples were examined by a microscope before they were put into the mass spectrometer. Needle-shaped crystal structures and inhomogeneous distributions of the samples were visible.
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Picomoles Flguro 4. Ion signals of 5'-AGTC3' vs the quantity of the oilgomer in the sample. Nlcotinic acid was used as matrix with laser fiuence of 30 mJ/cm2andlaser wavelengthof 266 nm. The quantlty of nicatkrlc acid in the sample was 120 nmol.
RESULTS AND DISCUSSION m i d negativebiopolymer ion spectra are shown in Figure 2 and Figure 3 for protein and oligonucleotide mixtures.
Measurement of a single-stranded 4-base oligonucleotide in a fixed quantity of matrix is shown in Figure 4. However, signals from the oligonucleotidesare not linearly proportional to the quantity of analyte. When the molar ratio is kept the same for matrix material to analyte, results of signal level vs analyte quantity are shown in Figure 5. This very nonlinear relationship between ion signal and analyte is mostly due to the number of layers of analyte the laser beam can probe. Increasing the fraction of analyte in the samples will not necessarily produce bigger signals. Figures 4 and 5 indicate that absolute quantitative measurements of biopolymersusing matrix-assisted laser desorption are extremely difficult since the signal level is strongly dependent on laser fluence, laser bean homogeneity, sample homogeneity,and substrate surface conditions. It is very difficult to keep these conditions the same throughout many measurements. Peak heights were used for plotting the data; however, little difference in the result is expected if peak areas were to be used for quantitative
Flguro 5. Negative ion signal of 5'-AGTC-3' vs the quantity of the oligomer. The matrix material used was nicotlnic acid. The molar ratio of maw to analyte was fixed at 10 000: 1. The laser fluence was 30 mJ/cm2 at a wavelength of 266 nm.
measurements. With a known quantity of biopolymershaving similar chemical properties used as an internal calibration, the signal level can be more or less independent of the above experimental variables, since inhomogeneous sample distribution on the substrate and the strong dependence of desorption on laser power should equally affect both the analyte and the material put into the sample for calibration. Figure 6 shows the experimental results of quantitative measurements of lysozyme and myoglobin using cytochrome
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ANALYTICAL CHEMISTRY, VOL. 65,NO. 15, AUGUST 1, 1993 2.50
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Figure 8. Ratlo of lysozyme and myoglobin to cytochrome c vs the true ratlo In the sample. Different slopes were obtained due to different detectlon efficlency. A good linear relatlonshlp Indicates Internal calibration Is a feaslble method for quantltathre measurements for blopolymers with similar chemical properties. The laser wavelength was 266 nm, with the laser fiuence at 75 mJ/cm2. Nlcotlnlc acM was used as matrix with the molar ratio of matrlx to cytochrome c at 10 0OO:l. The quantity of cytochrome c was fixed at 30 pmol.
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Molar Ratio to 2 mer Figwe 7. Signal ratlo of negative Ions of 3 mer (5'-AGT-3') and negative ions of 4 mer (5'-AGTC-3') to 2 mer (5'-AG3') vs molar ratios In the sample. A good linear relationshipwasobtalned. The laser wavelength was 266 nm with laser fluence at 30 mJ/cm2. Nicotinic acM was used as matrix with the molar ratio of matrix to 2 mer at 4000: 1. The quantity of 2 mer was fixed at 30 pmol.
c as an internal Calibration. A linear relationship was obtained between the molar ratio and signal ratio. Figure 7 shows quantitative results of small oligomers of 3 mer and 4 mer when 2 mer was used as the internal calibration. A good
Figure 8. Signal ratio of positive ion of cytochrome c to positive Ion of d(T)lo vs the molar ratio of cytochrome c to d(T)lo in the sample. The laser wavelength used was 355 nm with laser fluence at 50 ml/ om2. 2,5-DihydroxybenzoicacM was used as matrix material with the molar ratio of matrix to d(T)lo at 10 0OO:l.
linear relationship between signals and the quantity of analyte was also achieved. On the other hand, the results of signal ratios of protein using a poly-T oligomer as an internal calibration may not achieve a good linear relationship. Experimental results are shown in Figure 8. Plotted data points are the arithmetic mean of several samples, with error bars indicating f1standard deviation. These results indicate that biopolymers with different chemical properties should not be used for internal calibration. In conclusion, it is difficult to obtain absolute quantitative measurements of biopolymers using MALDI. However, internal calibration with molecules having similar chemical properties can be used to resolve these difficulties. Chemical reactions between biopolymers must be avoided to prevent the destruction of the analyte materials.
ACKNOWLEDGMENT We thank K. B. Jacobson, R. Haglund, M. J. Doktycz, and K. L. Lee for very valuable discussions. Research was sponsored by the Office of Health and Environmental Research, U.S.Department of Energy, under Contract DEAC05-840R21400with Martin Marietta Energy Systems, Inc. Preparation of the manuscript by Darlene Holt is also acknowledged.
RECEIVED for review November 20, 1993.
5, 1992. Accepted May
