Anal. Chem. 1999, 71, 4160-4165
The Dominance of Arginine-Containing Peptides in MALDI-Derived Tryptic Mass Fingerprints of Proteins Eberhard Krause,† Holger Wenschuh,‡ and Peter R. Jungblut*,§
Institute of Molecular Pharmacology, Alfred Kowalke-Str.4, D-10315 Berlin, Germany, Jerini Bio Tools GmbH, Rudower Chaussee 29, D-12489 Berlin, Germany, and Max-Planck-Institute for Infection Biology, Protein Analysis Unit, Monbijoustr. 2, D-10117 Berlin, Germany
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool for mass fingerprinting of peptide mixtures obtained after enzymatic ingel digestion of proteins separated by two-dimensional electrophoresis (2-DE). In the course of a proteome analysis of mycobacteria using mass spectrometric identification, it was found that 94% of the most intense MALDI-MS peaks denote peptides bearing arginine at the C-terminal end. The effect was demonstrated to be equally prominent using an equimolar mixture of the synthetic peptides known to be present in the tryptic digest of the mycobacterial 35 kDa antigen (“synthetic mass map”). In addition, several binary mixtures of synthetic peptides differing exclusively at the C terminus (Arg or Lys) were examined to rationalize the higher sensitivity toward arginine-containing peptides. The extent of the effect described depends on the matrix used and may facilitate a more reliable assignment of mass fingerprint data to protein sequences in databases. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become a powerful tool to identify proteins in large-scale proteome investigations. Enzymatic digestion of proteins in-gel or on-blot followed by MALDI-MS of the resulting peptide mixture leads to a peptide mass fingerprint, which is characteristic for the proteins present. Since the first description of peptide mass fingerprinting1-4 the method has been optimized5-8 and used for the identification of large numbers of proteins from * Corrresponding author. Fax: 49-30-280-26-627. E-mail: jungblut@ mpiib-berlin.mpg.de. † Institute of Molecular Pharmacology. ‡ Jerini Bio Tools GmbH. § Max-Planck-Institute for Infection Biology. (1) 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. (2) Pappin, D. J. C.; Hojrup, P.; Bleasby, A. J. Curr. Biol. 1993, 3, 327-332. (3) Mann, M.; Hojrup, P.; Roepstorff, P. Biol. Mass Spectrom. 1993, 22, 338345. (4) James, P.; Quadroni, M.; Carafoli, E.; Gonnet, G. Biochem. Biophys. Res. Commun. 1993, 195, 58-64. (5) Houthaeve, T.; Gausepohl, H.; Mann, M.; Ashman, K. FEBS Lett. 1995, 376, 91-94. (6) Otto, A.; Thiede, B.; Mu ¨ ller, E.-C.; Scheler, C.; Wittmann-Liebold, B.; Jungblut, P. Electrophoresis 1996, 17, 1643-1650. (7) Eckerskorn, C.; Strupat K.; Kellermann J.; Lottspeich F.; Hillenkamp F. J. Protein Chem. 1997, 16, 349-362.
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two-dimensional protein patterns.9,10 The sequence coverage obtained depends largely on the amount of protein available and the staining procedure.11 Additional external factors, such as sample preparation methods,12 matrix solution conditions,13 and matrix crystal morphology14-18 have been shown to affect the peak intensity of peptides. Although much work has been directed to the optimization of experimental conditions, peak intensities of peptides differ significantly, and a sequence coverage between 40 and 60% is typical. Recent results suggested that several intrinsic properties of peptides influence their MALDI behavior. Besides charged side chains,13,19 the presence of aromatic amino acids,20 peptide hydrophobicity,21 size,21 and the potential to form stable secondary structures22 have been reported to influence ion intensity. Furthermore, so-called suppression effects were observed in peptide mixtures such as those obtained by tryptic digestion.23 (8) Gevaert, K.; Demol, H.; Puype, M.; Broekaert, D.; De Boeck, S.; Houthaeve, T.; Vandekerckhove, J. Electrophoresis 1997, 18, 2950-2960. (9) Shevchenko, A.; Jensen, O. N.; Podteljnikov, A. V.; Sagliocco, F.; Wilm, M.; Vorm, O.; Mortensen, P.; Shevchenko, A.; Boucheri, H.; Mann, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14440-14445. (10) Mu ¨ ller, E.-C.; Thiede, B.; Zimny-Arndt, U.; Scheler, C.; Prehm, J.; Mu ¨ llerWerdan, U.; Wittmann-Liebold, B.; Otto, A.; Jungblut, P. Electrophoresis 1996, 17, 1700-1712. (11) Scheler, C.; Lamer, S.; Pan, Z.; Li, X.-P.; Salnikow, J.; Jungblut, P. Electrophoresis 1998, 19, 918-928. (12) Kussmann, M.; Nordhoff, E.; Rahbek-Nielsen, H.; Haebel, S.; Rossel-Larsen, M.; Jakobsen, L.; Gobom, J.; Mirgodskaya, E.; Kroll-Kristensen, A.; Palm, L.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 593-601. (13) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31-37. (14) Amado, F. M. L.; Domingues, P.; Santa-Marques, M. G.; Ferrer-Correia, A. J.; Tomer, K. B. Rapid Commun. Mass Spectrom. 1997, 11, 1347-1352. (15) Doktycz, S. J.; Savickas, P. J.; Krueger, D. A. Rapid Commun. Mass Spectrom. 1991, 5, 145-148. (16) Rosinke, B.; Strupat, K.; Hillenkamp, F.; Rosenbush, J.; Dencher, N.; Kru ¨ ger, U.; Galla, H. J. J. Mass Spectrom. 1995, 30, 1462-1468. (17) Chan, T. W. D.; Colburn, A. W.; Derrick, P. J.; Gardiner, D. J.; Browden, M. Org. Mass Spectrom. 1992, 27, 188-194. (18) Figueroa, I. D.; Torres, O.; Russell, D. H. Anal. Chem. 1998, 70, 45274533. (19) Zhu, Y. F.; Lee, K. L.; Tang, K.; Allman, S. L.; Taranenko, N. I.; Chen, C. H. Rapid Commun. Mass Spectrom. 1995, 9, 1315-1320. (20) Valero, M.-L.; Giralt, E.; Andreu, D. In Peptides 1996; Ramage R., Epton, R., Eds.; Mayflower Scientific Ltd.: Kingswinford, UK, 1998; pp 855-856. (21) Olumee, Z.; Sadeghi, M.; Tang, X. D.; Vertes, A. Rapid Commun. Mass Spectrom. 1995, 9, 744-752. (22) Wenschuh, H.; Halada, P.; Lamer, P.; Jungblut, P.; Krause, E. Rapid Commun. Mass Spectrom. 1998, 12, 115-119. (23) Kratzer, R.; Eckerskorn, C.; Karas, M.; Lottspeich, F. Electrophoresis 1998, 19, 1910-1919. 10.1021/ac990298f CCC: $18.00
© 1999 American Chemical Society Published on Web 08/28/1999
Figure 2. Amount of arginine- and lysine-containing peptides within the first five positions in the order of peak intensities. One hundred mycobacterial proteins were in-gel digested by trypsin and peptide mass fingerprints searched with the MS-FIT program in the NCBI sequence database. Identification of proteins with a sequence coverage below 30% were confirmed by PSD analysis.
Figure 1. Tryptic peptide mass fingerprint of 2-DE separated 35kDa antigen. A, Peptide mass fingerprint. The peaks of 35 kDa antigen are marked on the spectrum with their determined mass values. B, MS-FIT search results, showing the search criteria, sequence of the matching peptides, deviation of determined from theoretical mass value (delta), and the sequence coverage.
The present study examines the phenomenon that the presence of arginine has an influence on the peak intensity in tryptic mass fingerprints. The relation between the presence of the basic amino acids arginine and lysine in tryptic protein fragments and the appearance of the related ions in MALDI-MS fingerprints is studied on tryptic digests of numerous proteins and rationalized using several model systems. EXPERIMENTAL SECTION Sample Preparation. Mycobacterium tuberculosis strains H37Rv and Erdman, Mycobacterium bovis BCG Copenhagen and Chicago were grown in Middlebrook medium (500 mL, inoculum 5 × 107 to 1 × 108 bacteria) for 6 and 8 days, respectively, with continuous stirring. Cells were harvested by centrifugation (4000 rpm in a Heraeus tabletop centrifuge), and pellets were washed three times in phosphate-buffered saline (PBS) containing protease inhibitors (TLCK, Pepstatin A, E64, Leupeptin; 50 µM each). Pellets were ultrasonicated for 10 min at level 10, 50%, using a sonicator equipped with a head (Branson, USA), resuspended in 5 volumes of 9 M urea, 2% Triton X100, 25 mM Tris/HCl, pH 7.1, 50 mM KCl, 3 mM ethylenediaminotetraacetic acid disodium dihydrate (EDTA), 70 mM dithiothreitol (DTT), 2.9 mM benzamidine, 2.1 µM leupeptin, 0.1 µM pepstatin, 1 mM phenylmethylsulfonyl
fluoride (PMSF), and 2% carrier ampholytes (Servalyt, pH 2-4, Serva, Heidelberg, Germany), and incubated at room temperature for 30 min. Samples were centrifuged at 50 000 rpm (Beckman Optima TLX, TLA 100.4 rotor) for 30 min, and supernatants were collected. Protein concentration was determined by the method of Peterson.24 Two-Dimensional Electrophoresis. Two-dimensional electrophoresis (2-DE) was performed by a combination of carrier ampholyte urea isoelectric focusing (IEF) and SDS-PAGE25 in gels with a size of 23 × 30 cm2. The sample was applied at the anodic side of the IEF gel containing a pH gradient of 2-11 (Witalytes, 2-11, WITA, Teltow, Germany). IEF was run under nonequilibrium pH-gradient electrophoresis (NEPHGE) conditions (8870 Vh).25 SDS-PAGE was performed in 1.5-mm-thick 15% acrylamide gels using the IEF gel as the stacking gel. The proteins were detected by staining with Coomassie Brilliant Blue R250. Peptide Synthesis and Purification. Assembly of peptides was performed using standard solid-phase synthesis procedures by means of a MultiSynTech Syro II synthesizer (MultiSynTech GmbH, Witten, Germany). Initially, TG-S AC resin (loading, 0.24 mmol/g; Rapp-Polymere, Tu¨bingen, Germany) was loaded with the C-terminal Fmoc-amino acids (4 equiv.) via activation with diisopropylcarbodiimide (DIC) (4 eq.)/N-methylimidazole (NMI) (3 eq.) in dichloromethane (0.3M) for 3 h. Stepwise synthesis of the peptides was performed using 100 mg of the corresponding Fmoc-amino acid-TG SAC-resin for each peptide and coupling with 4 equiv. of Fmoc-amino acid/N-[(1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methyl-methanaminium tetrafluoroborate Noxide (TBTU) (4 equiv.)/diisopropylethylamine (DIEA) (8 equiv.) in dimethylformamide (DMF) (coupling concentration, 0.25 M; (24) Peterson, G. L. Anal. Biochem. 1977, 83, 346-356. (25) Klose, J.; Kobalz, U. Electrophoresis 1995, 16, 1034-1059.
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Figure 3. Comparison of synthetic peptide mass fingerprints from a 35-kDA antigen with six lysine-arginine exchanges. A, Peptide mass fingerprint of the synthetic peptide mixture with the original lysine-containing peptides. B, Peptide mass fingerprint of the synthetic peptide mixture with the arginine-containing peptides. C, Assignment of peaks to the peptide sequence and the peptide mass.
double coupling, 2 × 20 min). Finally, peptide-resin cleavage was performed with a solution of 2% triisopropylsilane, 5% water, 5% phenol in trifluoroacetic acid (TFA) for 2 h. After precipitation in tert-butyl methyl ether, peptides were purified by preparative HPLC using a Merck Hitachi D-7000 preparative HPLC system on a Merck Lichrosorb RP-18 (250 × 25 mm2 i.d., 7 µm) column. Peptides were eluted using a linear gradient of eluent A (0.1% TFA in water) and eluent B (0.1% TFA in 80% acetonitrile/20% water (v/v)). After lyophilization, all peptides were shown to have a purity >95% according to HPLC and consistent MALDI-TOFMS data (Voyager-DE, Biospectrometry Workstation, Perseptive Biosystems Inc., Framingham, MA). Amino acid analysis of purified peptides was performed using ion-exchange chromatography and postcolumn derivatization with ninhydrin (BiotronikEppendorf LC 3000) after hydrolysis in 6 N HCl at 120 °C for 48 h. Identification of Proteins by Peptide Mass Fingerprinting. After tryptic in-gel digestion in 100 mM Tris/HCl pH 8.0, 1 mM CaCl2, 10% (v/v) acetonitrile containing 1 µg of trypsin (Promega, 4162 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
Madison, WI) overnight at 36 °C, the resulting peptides were washed and concentrated within a peptide-collecting device26 and identified by peptide mass fingerprinting. Peptides were eluted from the collecting device in 100 µL of 60% acetonitrile, 0.1% TFA. Two microliters of this solution was mixed with 2 µL of a saturated R-cyano-4-hydroxy cinnamic acid (4-HCCA) solution in 50% acetonitrile, 0.3%TFA, and 2 µL was applied to the MALDI-MS sample slide. In the case of low-intensity spots, the eluate was concentrated up to 10-fold before mixing with the MALDI-MS matrix. A mass accuracy in the range of 30 ppm was obtained by double internal calibration with three peptides of known molecular mass using the Voyager Elite MALDI-mass spectrometer (Perseptive Biosystems, Inc.). Data were obtained from 256 laser shots using the following parameters: positive-ion reflector mode, 20 kV accelerating voltage, 70% grid voltage, 0.050% guide wire voltage, 100-ns delay, and a low mass gate of 500. Proteins were identified using the search program MS-FIT(http://falcon.ludwig.ucl.ac.uk/msfit.htm) reducing the proteins of the NCBInr11.22.97 database release to the Mycobacteria proteins and to a molecular mass range estimated from 2-DE ( 20%, allowing a mass accuracy of 0.1 Da. If there were no hits the molecular mass window was extended. Partial enzymatic cleavages leaving one cleavage site, oxidation of methionine, pyro-glutamic acid formation at N-terminal glutamine, and modification of cysteine by reaction with acrylamide were all considered in these searches. MALDI-MS Investigations of Synthetic Peptides. The mass spectrometry measurements were performed on a Voyager-DE STR BioSpectrometry Workstation MALDI-TOF mass spectrometer (Perseptive Biosystems, Inc.). Three matrix systems were used. A solution of 10 mg of matrix substance/milliliter of solvent (50% acetonitrile/0.3% TFA) was used with 4-HCCA and sinapinic acid, and 20 mg of matrix substance/milliliter of solvent (30% acetonitrile/0.3% TFA) was used with 2,5-dihydroxybenzoic acid (DHB). Although the “dried-droplet” technique, in which sample and matrix are premixed before spotting, was found to give somewhat lower signal intensities than the “thin layer” technique, it provided better reproduction of peak intensities from different spots.22 For this reason the “dried-droplet” technique was used throughout. The protocol was as follows: matrix solution (2 µL) was premixed with 2 µL of peptide solution (10 fmol/µL - 10 pmol/µL) dissolved in 5% acetonitrile/0.1% TFA. From the resulting mixture, 1 µL was applied to the sample plate. Samples were air-dried at ambient temperature (24 °C) using a cover-box for the sample slide to avoid differences in the crystallization process. All measurements were performed in the positive-ion reflection mode at an accelerating voltage of 20 kV and a delay of 200 ns. Each spectrum obtained was the mean of 256 laser shots. Reversed-Phase HPLC of Binary Mixtures of Peptides. Chromatographic measurements were performed on a Jasco gradient HPLC system (Jasco GmbH, Germany) consisting of two PU-980 pumps, an AS-950 autoinjector, and a UV-975 detector operating at 220 nm. Runs were carried out on a Vydac C18 column (250 × 4.6 mm i.d., 5 µm, Vydac, Inc., Hesperia, CA). Mobile phase A was 0.1% TFA in water and B was 0.1% TFA in acetonitrile-water (80:20, v/v). The sample concentration was 0.5 (26) Otto, A.; Thiede, B.; Mu ¨ ller, E.-C.; Scheler, C.; Wittmann-Liebold, B.; Jungblut, P. Electrophoresis 1996, 17, 1643-1650.
mg/mL of each peptide in eluent A, and the injection volume was 20 µL. Separations were performed at 22 °C using a linear gradient of 5-50% B in 40 min at an eluent flow rate of 1 mL/min. Electrospray Mass Spectrometry. Electrospray Ionization (ESI) mass spectrometry of binary mixtures of peptides was performed on a triple-quadrupole instrument (TSQ 700, Finnigan MAT, Bremen, Germany) equipped with an electrospray ion source (API-ESI) operating in the positive mode with a capillary temperature of 200 °C and a voltage of 4.5 kV. The sample concentration was 5 pmol/µL in methanol-water-acetic acid (50: 49:1, v/v/v). The samples were introduced into the ion source at a constant flow rate of 3 µL/min. Nitrogen was used as sheath gas at a pressure of 3.4 bar. The spectra were the averaged sum of 64 scans. RESULTS AND DISCUSSION Peptide Mass Fingerprinting from 2-DE Separated Proteins. One hundred proteins from Mycobacteria species separated by 2-DE and in-gel digested by trypsin were investigated by MALDI-MS using 4-HCCA as matrix. Upon evaluating the most intense peaks in the mass range from 500-3000 Da, it was found that a large majority could be assigned to peptides bearing arginine at the C terminus. Figure 1a displays the peptide mass fingerprint of the Mycobacterium tuberculosis 35-kDa protein. Clearly, peptides matching the most intense peaks contain arginine at the C-terminal position (Figure 1b). Comparison of the mass fingerprints of all proteins with regard to the abundance of either arginine or lysine in the five most intense peaks of the spectrum revealed that peaks of peptides containing arginine at the C terminus are more prominent (Figure 2). The data show that 94% of the most intense peaks detected were related to peptides bearing arginine in the sequence. Although the percentage of C-terminal lysine-containing peptides increased slowly from position 1 to 5 (6% to 28%, respectively), the preponderance of arginine-containing peptides is clearly demonstrated even at position 5 on the intensity scale. Further analysis of peptides having a C-terminal lysine and appearing at position 1 revealed that 67% of them contained at least one additional basic amino acid. Hydrophobicity and pI calculated from the peptide sequence at the first five positions did not reveal any correlation with peak intensity. Interestingly, a slightly higher number of peptides containing phenylalanine was observed in the most intense peak as compared with peptides with lower peak intensities, thus confirming a peak-enhancing effect from better desorption of aromatic amino acids from the template as described earlier.20 The effect results in MALDI mass fingerprints of tryptic protein digests which are dominated by arginine-containing peptides. These observations might be caused by (i) differences in enzyme kinetics, i.e., a more effective cleavage after arginine, (ii) molecular interactions between Lys- and Arg-containing peptides in mixtures leading to a relative suppression of peak intensities of Lys peptides compared with those corresponding to Arg peptides, (iii) peakenhancing effects related to the specific chemical nature of arginine residues. To clarify the situation, synthetic mixtures of well-characterized peptides were investigated. Fingerprint of 35 kD Protein Synthetic Peptides. Eighteen tryptic peptides of M. tuberculosis 35kD protein in the mass range 500-3000 were synthesized, purified to homogeneity, and ana-
Table 1. Influence of the C-terminal Basic Amino Acid (Lys/Arg) on the Relative Peak Intensity of Peptide Fragments in Synthetic Mass Fingerprints (18 Peptides) of M. tuberculosis 35-kDa Antigena amino acid sequence 1-7 11-19 20-27 65-71 140-147 154-162 1-7 11-19 20-27 65-71 140-147 154-162
amount (pmol)
relative peak intensity with K at the C terminus
relative peak intensity with R at the C terminus
R/K intensity factor
0.5 0.5 0.5 0.5 0.5 0.5 10 10 10 10 10 10
0.08 0.15 n.d. n.d. n.d. 0.03 0.08 0.35 n.d. n.d. 0.04 0.06
0.35 0.62 0.08 0.07 0.16 0.55 0.65 1.00 0.10 0.22 0.32 0.65
4.4 4.1 >4.0 >4.0 >4.0 18.3 8.1 2.9 >4.0 >4.0 8.0 10.8
a The peptide mixture was analyzed by MALDI-MS using 4-HCCA matrix. b n.d., not detectable.
lyzed by analytical HPLC and mass spectrometry. The peptides were mixed in equimolar amounts to provide a “synthetic mass fingerprint” and analyzed by MALDI-MS using different matrix systems. A comparison of the spectrum of the tryptic protein digest (Figure 1) with that of the synthetic peptide mixture (Figure 3A) containing 500 fmol reveals that corresponding peaks have roughly the same peak intensity. As shown in Figure 3A, the most intense peaks of the synthetic mixture are, again, argininecontaining peptides. Remarkably, all arginine-containing peptides were detected, whereas 3 lysine-containing peptides are missing. Additional measurements were performed on a mixture in which the C-terminal amino acid lysine of 6 peptides (peptides 4, 5, 6, 7, 8, 9 in Figure 3C) was replaced by arginine. Figure 3B shows that the exchange of a terminal lysine for arginine clearly increased the relative peak intensity. Table 1 summarizes the results obtained using 4-HCCA as matrix. The exchange of amino acids resulted in a 4- to 18-fold increase of peak intensity. Using DHB and sinapinic acid the increase of peak intensity as a result of the Lys f Arg exchange was determined to be 2- and 3.2-fold, respectively. These well-characterized synthetic mass fingerprints show that the preference of finding arginine-containing peptides in the MALDI-MS spectra of tryptic digests is not caused by an incomplete enzymatic digestion of Lys- vs Arg-containing peptides. Binary Mixtures of 35-kDa Protein Peptides with LysineArginine Exchanges. To study the differences in peptide-ion intensity in detail, equimolar binary mixtures of peptides containing arginine or lysine at the C-terminal end were investigated. The corresponding arginine peptides were fragments of the 35kDa protein described. These binary mixtures were analyzed by HPLC, ESI-MS, and MALDI-MS. Spectra of the peptides MANPFVK and MANPFVR mixed in equimolar amounts are shown in Figure 4. The results, summarized in Table 2, show that all Arg/ Lys peptide mixtures investigated gave intensity factors (IArg peptide/ ILys peptide) of approximately 1 in reversed-phase HPLC and ESIMS. In contrast, the MALDI-MS spectra confirm the tremendous differences in signal intensities observed for multiple peptide mixtures. In comparison to a terminal lysine, the presence of Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
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Figure 4. Peak intensity comparison of an equimolar binary mixture of MANPFVK/R in HPLC, ESI-MS, and MALDI-MS. A, HPLC; B; ESI-MS, the double protonated peptide peaks are shown; C, MALDI-MS. The peak at 850.271 represents a methionine-oxidized form of the Arg peptide. Table 2. Peak Intensity Relationship of Binary Mixtures of C-Terminally Exchanged R to K Peptides (Sequences of M. tuberculosis H37Rv 35-kDa Antigen) Measured by HPLC (1 nmol), ESI-MS (1 pmol/µL), and MALDI-MS (0.5 pmol, 4-HCCA Matrix) intensity factor R/K peptide
position
HPLC
ESI-MS
MALDI-MS
MANPFVR/K YLMALFSSR/K IDEHADPR/K QLADIER/K NAMVLQQR/K LLSQLEQAR/K
1-7 11-19 20-27 65-71 140-147 154-162
1.09 1.03 1.04 1.17 1.11 1.02
1.23 1.01 1.02 1.06 1.14 0.84
5.19 4.54 10.8 13.25 15.29 4.46
Table 3. MALDI-MS Peak Detection Limit of Peptides with K or R at the C-Terminal Position, Specifically LLSQLEQAR/K (Fragment 154-162) and MANPFVR/K (Fragment 1-7) of M. tuberculosis 35-kDa Antigen Were Analyzed Using 4-HCCA as Matrix peptide amount (fmol)
K 154-162 counts
R 154-162 counts
K 1-7 counts
R 1-7 counts
500 250 100 50 10
33 000 4000 n.d. n.d. n.d.
34 000 22 000 9000 2300 n.d.a
28 000 1200 n.d. n.d. n.d.
33 000 16 000 9500 12 500 n.d.
a
arginine results in 4.5- to 15.3-fold higher peak intensities, which is in agreement with the “arginine effect” measured with the mass fingerprints (4- to 18-fold). Determination of Signal Intensities and Detection Limits of Single Arginine and Lysine Peptides. In an attempt to elucidate the nature of the different MALDI-MS behaviors of lysine-containing vs arginine-containing peptides in mixtures, measurements of single peptides were performed. The synthetic peptide fragments 154-162 and 1-7 of the 35-kDa protein with either lysine or arginine at the C terminus were measured at various concentrations from 50 to 500 fmol. Similarly as in the peptide mixtures, the Arg-containing peptides gave higher intensities than their lysine counterparts when measured on separated samples. At 250 fmol the Arg-containing peptides showed 5.5 to 13-fold higher intensities than their lysine counterparts. At 500 fmol the difference was reduced, but still present. Reduction of peptide amount to the 100- and 50-fmol level led to the disappearance of signals in the case of the Lys peptides, whereas peptides containing Arg could still be detected (Table 3). (27) Harrison, A. G. Mass Spectrom. Rev. 1997, 16, 201-217. (28) Li, X. P.; Harrison, A. G. Org. Mass Spectrom. 1993, 28, 366-371.
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n.d. not detectable.
These results clearly indicate that the observed differences in signal intensity in peptidic mixtures are not related to any effect resulting from molecular interactions between components in peptide mixtures leading to suppressed Lys-peptide signals as described for other tryptic mixtures.23 The effect found is rather ascribed to the presence of the arginine moiety in the studied peptides. This is in agreement with earlier contributions which addressed the enhancement of signal intensities by the presence of basic amino acids in the peptide chain resulting from an acidbase reaction between protein/peptide and the matrix.19 The described liquid-phase protonation mechanism results in a lower energy barrier for the ionization in the MALDI process for peptides bearing basic amino acids and may lead to the higher signals observed. In addition, the effect might further be attributed (29) Bojesen, G.; Breindahl, T. J. Chem. Soc., Perkin Trans. 1 1994, 2, 10291037. (30) Wu, Z. C.; Fenselau, C. Rapid Commun. Mass Spectrom. 1994, 8, 777780. (31) Wu, J. Y.; Lebrilla, C. B. J. Am. Chem. Soc. 1993, 115, 3270-3275. (32) Wu, Z. C.; Fenselau, C. J. Am. Soc. Mass Spectrom. 1992, 3, 863-866. (33) Zhang, K.; Zimmerman, D. M.; Chung-Phillips, A.; Cassady, C. J. J. Am. Chem. Soc. 1993, 115, 10812-10822.
to the high gas-phase basicity and proton affinity of arginine in comparison with those of other proteinogenic amino acids including lysine, as reviewed by Harrison27 and pointed out by others,28-33 deriving from a preferred protonation of the Arg peptides. However, from the present data no conclusive mechanism can be established. Details regarding this matter must await further studies. CONCLUSION On examining MALDI-MS ion intensities of tryptic peptide maps from 100 proteins, it was found that the peak intensities of arginine-containing peptides are significantly higher than those of lysine peptides. Experiments on synthetic mixtures of peptides known to be present in a tryptic digest as well as single peptide measurements confirm that the phenomenon is related to the specific chemical properties of arginine. The outstanding basicity of the guanidino functionality of the arginine side chain may result in preferred ionization in the liquid and/or gas phase, yielding
higher ion intensities from arginine peptides than from those which do not contain arginine. The detected general dominance of arginine-containing peptides in MALDI-MS spectra of tryptic protein digests has to be taken into account in order to rationalize incomplete mass maps and provides an additional criterion for the reliable assignment of a peptide mass fingerprint to a protein in database searches. The assignment of a protein spot in a 2-DE gel to a protein is confirmed if the most intense peaks belong to Arg-containing peptides. ACKNOWLEDGMENT The authors thank S. Lamer, M. Weissbach, and H. Lerch for excellent technical assistance. Received for review March 18, 1999. Accepted July 19, 1999. AC990298F
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