A Calibration Method That Simplifies and Improves Accurate

Anal. Chem. 2002, 74, 3915-3923

A Calibration Method That Simplifies and Improves Accurate Determination of Peptide Molecular Masses by MALDI-TOF MS Johan Gobom,*,† Martin Mueller,‡ Volker Egelhofer,† Dorothea Theiss,† Hans Lehrach,† and Eckhard Nordhoff†,‡

Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany, and Scienion AG, Volmerstrasse 7a, 12489 Berlin, Germany

The use of delayed ion extraction in MALDI time-of-flight mass spectrometry distorts the linear relationship between m/z and the square of the ion flight time (t2) with the consequence that, if a mass accuracy of 10 ppm or better is to be obtained, the calibrant signals have to fall close to the analyte signals. If this is not possible, systematic errors arise. To eliminate these, a higher-order calibration function and thus several calibrant signals are required. For internal calibration, however, this approach is limited by signal suppression effects and the increasing chance of the calibrant signals overlapping with analyte signals. If instead the calibrants are prepared separately, this problem is replaced by an other; i.e., the ion flight times are dependent on the sample plate position. For this reason, even if the calibrants are placed close to the sample, the mass accuracy is not improved when a higherorder calibration function is applied. We have studied this phenomenon and found that the relative errors, which result when moving from one sample to the next, are directly proportional to m/z. Based on this observation, we developed a two-step calibration method, that overcomes said limitations. The first step is an external calibration with a high-order polynomial function used for the determination of the relation between m/z and t2, and the second step is a first-order internal correction for sample position-dependent errors. Applying this method, for instance, to a mass spectrum of a mixture of 18 peptides from a tryptic digest of a recombinant protein resulted in an average mass error of 1.0 ppm with a standard deviation of 3.5 ppm. When instead using a conventional two-point internal calibration, the average relative error was 2.2 ppm with a standard deviation of 15 ppm. The new method is described and its performance is demonstrated with examples relevant to proteome research. Calibration in matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF MS) is the conversion * Corresponding author. Phone: +49 30 84131542. Fax: +49 30 84131139. E-mail: [email protected]. † Max Planck Institute for Molecular Genetics. ‡ Scienion AG. 10.1021/ac011203o CCC: $22.00 Published on Web 06/22/2002

© 2002 American Chemical Society

of a recorded time-of-flight spectrum to a mass spectrum. This conversion is usually achieved by measuring the flight times t of at least two ions of known masses (calibrants) and determining the calibration constants, a0 and a1, in the equation

m/z ) a0 + a1t2

(1)

This equation is then used to determine unknown masses. The calibrants are prepared either together with the sample of interest (internal calibration) or separately on another position of the sample support (external calibration). Equation 1 is a first-order approximation that, for example, does not take into account the initial velocity and energy deficit of the ions. It was shown that addition of a third term in the calibration equation resulted in improved mass accuracy for linear TOF mass analyzers.1 The performance of MALDI-TOF mass spectrometers was greatly improved by the implementation of time-delayed ion extraction,2-5 based on the work of Wiley and McLaren.6 For the routine analysis of peptides, this technique has raised the signal resolving power from 2000 to beyond 10 000 (fwhm) and has improved the limit of detection by a similar order. As a consequence of improved signal resolution and signal-to-noise ratio, the accuracy of peptide molecular mass determinations has also been improved, with 5-10 ppm as a typical value if calibrants spanning a narrow mass range and bracketing the ions of interest are chosen.7,8 If a broader mass window (e.g., 700-4000 Da) is calibrated using two reference signals, the maximum relative error, however, can increase considerably (30-50 ppm). One reason for this is that time-delayed ion extraction results in deviations from linearity of m/z versus t2.9 The actual correlation depends on many parameters including the ions’ initial velocity, (1) Vera, C. C.; Zubarev, R.; Ehring, H.; Hakansson, P.; Sunqvist, B. U. R. Rapid Commun. Mass Spectrom. 1996, 10, 1429-1432. (2) Colby, S. M.; King, T. B.; Reilly, J. P. Rapid Commun. Mass Spectrom. 1994, 8, 865-868. (3) Whittal, R. M.; Li, L. Anal. Chem. 1995, 67, 1950-1954. (4) Brown, R. S.; Lennon, J. J. Anal. Chem. 1995, 67, 1998-2003. (5) Vestal, M. L.; Juhasz, P.; Martin, S. A. Rapid Commun. Mass Spectrom. 1995, 9, 1044-1050. (6) Wiley, W. C.; McLaren, I. H. Rev. Sci. Instrum. 1955, 26, 1150. (7) Edmondson, R. D.; Russell, D. H. J. Am. Soc. Mass Spectrom. 1996, 7, 995-1001. (8) Takach, E. J.; Hines, W. M.; Patterson, D. H.; Juhasz, P.; Falick, A. M.; Vestal, M. L.; Martin, S. A. J Protein Chem 1997, 16, 363-369.

Analytical Chemistry, Vol. 74, No. 15, August 1, 2002 3915

experimental conditions, and instrument design.10 Accounting for the influence of these parameters requires the use of a higherorder calibration equation, which implies either the use of multiple calibrants or predetermination of some calibration constants based on knowledge of relevant instrumental parameters. For internal calibration, the former approach is impractical due to signal suppression in MALDI and the risk that the calibrant signals overlap with the signals of the ions of interest. By the latter approach, mass determinations with errors as low as 5 ppm over the m/z range of 900-3700 were demonstrated possible using a third-order calibration equation in which two constants relating to instrumental parameters and ion initial velocity were predetermined.11 Increasing the number of terms in the calibration equation evidently decreases the error but requires knowledge of more instrument parameters, which may be difficult to establish. In addition to the complex influence of instrumental parameters, the flight times in MALDI-TOF MS are also sample positiondependent, mainly because of imperfections in the sample plate planarity. Even if the calibrants are prepared close to the analyte, the respective systematic error can exceed 50 ppm although the distance of the sample to the first extraction electrode is shortened or extended only by a few micrometers. Mainly for this reason, a better mass accuracy is obtained by internal calibration of the recorded flight times. We found that the relative mass errors, which result from changing the sample position, correlate linearly to m/z and can therefore be eliminated by a first-order correction. Based on this observation, we developed a two-step calibration method, which separates the determination of an appropriate calibration function from the internal correction of sample position-dependent errors. For determination of the calibration function, a polymeric mixture, covering the entire mass range of interest (700-4000 Da), is analyzed on one position of the sample support. A curve-fitting algorithm is then used to determine the calibration constants. Because of the large number of detected signals, a high polynomial order can be used so that the function closely follows all systematic trends. This determination is entirely empirical and requires no a priori knowledge of the parameters that affect the linearity of the correlation of m/z versus t2. The determined constants are used to transform the recorded flight times to m/z values for all other samples on the support acquired under the same experimental conditions. Two internal reference masses are then used for a first-order correction of the sample positiondependent errors. We show that the proposed calibration routine is independent of the instrument’s actual correlation between m/z and t2 and thereby allows tuning the instrument for optimal detection sensitivity. The remaining relative mass errors obtained for routine analyses (automatic acquisition) are in the low-ppm range. MATERIALS AND METHODS Materials. The following three poly(propylene glycol) (PPG) oligomer fractions were purchased from Aldrich (Milwaukee, WI): Mn 1000 (20,232-0), Mn 2000 (20,233-9), and Mn 2700 (20,234-7). (9) Juhasz, P.; Roskey, M. T.; Smirnov, I. P.; Haff, L. A.; Vestal, M. L.; Martin, S. A. Anal. Chem. 1996, 68, 941-946. (10) Vestal, M.; Juhasz, P. J. Am. Soc. Mass Spectrom. 1998, 9, 892-911. (11) Juhasz, P.; Vestal, M. L.; Martin, S. A. J. Am. Soc. Mass Spectrom. 1997, 8, 209-217.

3916

Analytical Chemistry, Vol. 74, No. 15, August 1, 2002

The peptides angiotensin I and II (human), substance P-methyl ester, neurotensin 1-11 and 1-13, ACTH 1-17 and 18-39, and somatostatin 28 (human) were purchased from Bachem. R-Cyano4-hydroxycinnamic acid (CHCA) was purchased from Sigma (St. Louis, MO). The recombinant protein, donated by Dr. Harald Seitz at Max Planck Institute for Molecular Geneteics Berlin, was selected from a human fetal brain expression library. Preparation of Calibrants. The three PPG fractions were diluted 1:10 000 (v/v) in 99% acetone, 0.001% TFA (v/v). The three fractions were mixed in the ratio 1:2:3 (Mn 1000/2000/2700) (v/ v), aliquoted, and stored at -20 °C prior to use. The MALDI matrix solution was prepared by ultrasonicating an excess of CHCA in 99% acetone, 0.001% trifluoroacetic acid (TFA) (v/v) for 1 min. The matrix solution was mixed with the PPG calibrant mixture at a ratio of 4:1 (v/v). A few sodium chloride crystals were added to the solution to enhance sodium cationization of the PPG molecules. A volume of the solution was aspirated by capillary force into a narrow pipet tip (GELoader, Eppendorff). MALDI samples of the calibrant mixture were prepared by touching the hydrophilic sample anchors of a Scout MTP prestructured sample support (Scout 384-MTP AnchorChip, Bruker Daltonik, Bremen, Germany), with the outlet of the pipet tip whereby a small volume of the PPG/matrix solution was deposited. Peptide Sample Preparation. All peptide samples were prepared using the CHCA surface affinity preparation, previously described.12 This procedure yields homogeneous microcrystalline preparation, which minimizes signal shifts when summing up single-shot spectra from different positions on a sample spot. MALDI-TOF Spectra Acquisition. All mass spectra were acquired on a Bruker Reflex III Scout MTP instrument. Positively charged ions were analyzed in the reflector mode, using delayed ion extraction. Spectra were recorded with a 2-GHz data-sampling rate. Unless otherwise stated, the extraction delay time was 150 ns and deflection was used to suppress ions up to m/z 500. Other instrument parameters were tuned for optimal resolution (∼13 000, fwhm) around m/z 2500, resulting in resolutions of ∼5000 at m/z 700 and 4000. Recorded spectra of the PPG calibrant mixture were the sum of 400 single-shot spectra. For each peptide sample, 200 single-shot spectra were accumulated. All instrument high voltages were left on between all analyses to ensure a stable instrument performance. After short interruptions (1 h and (b) 7 min.

dropped to increasingly negative values. The average error was -264 ppm after 120 min and decreased to -286 ppm 40 min later. When the sample was measured the following day (leaving all voltages on overnight), the average error had stabilized at -306 ppm. The average error measured 1 h later was -305 ppm. Exchanging the MALDI sample plate requires turning off the instrument high voltages for 7 min on the instrument used. To evaluate how this interruption affects the calibration, the sample plate from the previous experiment was ejected from the instrument and then immediately reinserted. A mass spectrum of a PPG sample was recorded directly after switching on the instrument high voltages and subsequently over a time period. A polynomial calibration was performed for the first spectrum and applied to the other spectra. The resulting relative mass error plots are shown in Figure 4b. Similarly to the previous example, the relative mass errors initially increased to 14 ppm after the first 10 min. Subsequently, the average error decreased to -15 ppm after 30 min. After 50 min, the error was stabile at -28 ppm. The same value was obtained when the sample was measured 20 min later. On the basis of these observations, we decided to leave all instrument high voltages on between analyses. The need to wait 3920

Analytical Chemistry, Vol. 74, No. 15, August 1, 2002

after inserting a new sample plate increases the analysis time significantly. Because the change of the relative error as a function of m/z between 30 and 70 min consisted mainly of an offset, a 30-min period for stabilization of the instrument voltages was sufficient when internal first-order correction was used. For external calibration, the voltages were switched on for minimum 50 min prior to spectra acquisition. Calibration of Peptide Time-of-Flight Spectra. The performance of the calibration procedure for peptide samples was evaluated using a mixture containing eight peptides. The mass spectrum shown in Figure 5a was acquired at sample plate position M17. The relative error plot after external polynomial calibration calculated for a PPG mass spectrum acquired at position I13 is shown in Figure 5b. The average error was -12 ppm with a SD of 14 ppm. The error plot after an external two-point calibration, using the PPG polymers of m/z 1085.753 and 3175.260 as calibrants, is shown for comparison. This calibration yielded a similar average error of -14 ppm with a SD of 15 ppm. A firstorder internal correction, using angiotensin II (m/z 1045.541) and somatostatin 28 (m/z 3147.471) as reference signals, of the spectrum externally calibrated with the polynomial function resulted in an average error of -0.13 ppm with a SD of 2.2 ppm (Figure 5c). In comparison, a two-point internal calibration of the spectrum, using the same reference signals, yielded an average error of -9.4 ppm with a SD of 14 ppm. An example of a more complex peptide mixture is shown in Figure 6. Out of the 44 assigned signals in the mass spectrum, acquired from a tryptic digest of recombinant human fascin (SwissProt Q16658) expressed in Escherichia coli, 18 matched the calculated masses of expected tryptic cleavage products (Figure 6a). The relative mass error plots after external polynomial calibration and external two-point calibration are shown in Figure 6b. A first-order internal correction of the spectrum externally calibrated with the polynomial function, using the peptides of m/z 1113.544 and 2184.075 as reference signals (Figure 6c), resulted in an average mass error of 1.0 ppm with a SD of 3.5 ppm. A two-point internal calibration, using the same peptides as calibrants, resulted in an average error of 2.2 ppm with a SD of 15 ppm. Figure 7a shows the tryptic peptide map of a protein sample from Torpedo californica, isolated by two-dimensional gel electrophoresis. A total of 104 signals were assigned in the spectrum using the SNAP algorithm. External polynomial calibration was performed followed by a first-order internal correction using the porcine trypsin autoproteolysis signals of m/z 842.509 and 2211.104, labeled c1 and c2, respectively, in the figure. All chordata protein sequences contained in the NCBI database (release, 21 October 2001) were searched using the program ProFound (Proteometrics.com). Other search parameters were as follows: maximum possible protein mass is 3000 kDa, cysteine residues are carbamidomethylated, methionine residues can be oxidized, and a maximum error of 50 ppm is allowed. The search identified the protein as ATP-synthetase β subunit. The relative error plot for the 23 matching masses after the two-step calibration is shown in Figure 7b. The relative errors observed for a two-point internal calibration using the same reference masses are plotted for comparison. The average relative error for the matching peptides after external polynomial calibration followed by a first-order

Figure 5. (a) Mass spectrum of a peptide mixture containing 25 fmol of (1) angiotensin I, (2) angiotensin II, (3) substance P-methyl ester, (4) neurotensin 1-11, and (5) neurotensin 1-13 and 50 fmol of (6) ACTH 1-17, (7) ACTH 18-39, and (8) somatostatin 89. (b) Relative error plots of the determined m/z values after external twopoint calibration (green circles) and after external polynomial calibration (blue squares). (c) Relative mass error plots of the same spectrum after external polynomial calibration followed by a first-order internal correction (blue squares). The relative mass errors of the same spectrum after a two-point internal calibration, using the same reference signals as calibrants, are plotted for comparison (green circles).

Figure 6. (a) Mass spectrum of a tryptic digest of recombinant human fascin (SwissProt Q16658) expressed in E. coli. Labeled signals that matched expected m/z values of the protein are green. Other labeled signals are red. (b) Relative error plots of the matching signals external two-point calibration (green circles) and after external polynomial calibration (blue squares). (c) The relative error plot of the same spectrum after external polynomial calibration, followed by a first-order internal correction, using the calculated m/z values of two peptide signals (m/z 1113.544 and 2184.076) as reference values, is shown in blue squares. The relative error plot of the same spectrum after a two-point internal calibration, using the same reference signals as calibrants is shown for comparison (green circles).

internal correction was 2.0 ppm with a SD of 8.7 ppm. With a two-point internal calibration, the average relative error was 12 ppm with a SD of 17 ppm. The two-step calibration thus yielded a significantly higher mass accuracy compared to the internal twopoint calibration. In this example, however, the SD was larger

than that observed for the peptide mixture shown in Figure 5. This is most likely caused by the greatly varying signal-to-noise ratio in the mass spectrum of the tryptic peptide map of ATPsynthetase β subunit. For instance, for the signal of m/z 1262.641, magnified in Figure 7c, interference with chemical noise limited Analytical Chemistry, Vol. 74, No. 15, August 1, 2002

3921

Figure 7. (a) Mass spectrum recorded from a tryptic digest of a human protein, isolated from a membrane preparation by two-dimensional gel electrophoresis. The protein was identified as ATP-synthetase β chain by 21 peptide signals (green), matching the protein with a relative error below 50 ppm. Labeled signals, which did not match the identified protein, are red. (b) The relative error plot for the matching signals after external polynomial calibration followed by a first-order internal correction is shown in blue squares. The trypsin autoproteolysis products of m/z 842.5100 (c1) and 2211.104 (c2) were used for the first-order internal correction. The relative error plot for the same matching signals after a two-point internal calibration, using the same trypsin autoproteolysis products as calibrants, is shown in green circles. (c) Enlargement of the signal of m/z 1262.461, which matched the identified protein with a relative mass error of -17 ppm. (d) Enlargement of the signal of m/z 1617.8048, which matched the identified protein with a relative mass error of -17 ppm.

the accuracy of the peak assignment. Another possibility, likely to occur in complex mixtures, is partially overlapping signals. An example of this is shown in Figure 7d. Protein Identification without Internal Calibration. For protein identification by peptide mass fingerprinting,13-17 the internal correction can be omitted, when the protein identification program MSA developed in our laboratory is used.18 This omission facilitates automated analyses, because only one calibration equation needs to be generated for all samples on the same (13) Mann, M.; Hojrup, P.; Roepstorff, P. Biol. Mass Spectrom. 1993, 22, 338345. (14) James, P.; Quadroni, M.; Carafoli, E.; Gonnet, G. Biochem. Biophys. Res. Commun. 1993, 1, 58-64. (15) Pappin, D. J. C.; Hojrup, P.; Bleasby, A. J. Curr. Biol. 1993, 3, 327-332. (16) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5011-5015. (17) Yates, J. R., III; Speicher, S.; Griffin, P.; Hunkapiller, T. Anal. Biochem. 1993, 214, 397-408. (18) Egelhofer, V.; Bu ¨ ssow, K.; Luebbert, C.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2000, 72, 2741-2750.

3922

Analytical Chemistry, Vol. 74, No. 15, August 1, 2002

MALDI sample support and the presence of two reference signals in each sample is not required. In contrast to other search engines that rely on the accuracy of the determined peptide masses from a protein digest as the main parameter to retrieve the correct protein from a sequence database, MSA uses the observed m/z values only to narrow the search down to a few hundred potential candidate sequences, a task for which the moderate mass accuracy obtained by external calibration is sufficient. For each of these candidate sequences, the program computes the relative deviations of the measured m/z values to their corresponding matches in the database. Linear regression analysis of the differences versus the calculated peptide m/z values are then performed for all candidate sequences and the standard deviation of each regression is used as the main parameter to identify the correct protein. This analysis is the equivalent of initially assuming that each protein is potentially the correct candidate and then plotting the error curves for all candidates. The correct protein can be singled out because it generates the most linear error curve. Thus,

MSA does not rely on a high mass accuracy for protein identification but on the observation that the relative mass errors for the correct protein candidate correlate linearly to m/z. This strategy for protein identification is part of a separate paper.19 CONCLUSION The mass accuracy for MALDI-TOF MS of peptides can be improved by the combined use of an external polynomial calibration and a first-order internal correction. The external calibration is performed by fitting the square of the observed flight times of the components of a polymeric mixture, which covers the entire mass range of interest, to their expected m/z values. Using a high polynomial order (e.g., 15) ensures that the calibration accounts for all perturbations from linearity of m/z versus t2. The internal correction utilizes two reference masses to correct for sample position-dependent relative errors, which were shown to correlate linearly to m/z. The improved mass accuracy achieved by this method was illustrated for the analysis of a standard peptide mixture and for proteolytic digests of proteins. The proposed calibration method should also be applicable to analytes other than peptides, for example, synthetic polymers. (19) Egelhofer, V.; Gobom, J.; Seitz, H.; Giavalisco, P.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2002, 74, 1760-1771

The errors that remain when our calibration procedure was used were observed to be not entirely random. This can, for example, be seen in the error plots shown in Figure 3. The residual systematic error appears not to be sample positiondependent; i.e., it was also observed for multiple spectra recorded from the same sample spot. This observation suggests that the mass accuracy for routine peptide analysis by MALDI-TOF MS is currently not limited by the data-sampling rate of the instrument (2 GHz was used in this study) but rather by the instability of critical instrument parameters, e.g., the electronics used for delayed ion extraction. ACKNOWLEDGMENT The authors thank E. Mirgorodskaya, K. D. Kloeppel, P. Giavalisco, Niklas Gustavsson, and E. Wolski at the Max Planck Institute for Molecular Genetics, Berlin, for support of our work and scientific discussion. This work was funded by the German Ministry for Education and Research (BMBF Project 31P2715) and the Max Planck Society. Received for review November 20, 2001. Accepted May 2, 2002. AC011203O

Analytical Chemistry, Vol. 74, No. 15, August 1, 2002

3923