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Characterization of Protein Biomarkers Desorbed by MALDI from Whole Fungal Cells Bijan Amiri-Eliasi* and Catherine Fenselau
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742
In this publication, the use of ultraviolet (UV) matrixassisted laser desorption/ionization (MALDI) time-offlight (TOF) mass spectrometry (MS) for rapid identification and characterization of Saccharomyces cerevisiae, a fungus, is reported. S. cerevisiae is a unicellular eukaroyte that can serve as a model to study more complex organisms. We have determined that the best technique for cell wall lyses for MALDI involves the use of high concentrations of formic acid solutions. We also have shown that different fungal species exhibit different mass spectra, which can be used to distinguish them readily. Protein peaks from S. cerevisiae spectra have been tentatively identified using bioinformatics and are mainly assigned to ribosomal and mitochondrion-related proteins. Since its introduction in the late 1980s,1,2 matrix-assisted laserdesorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has found application in many areas.3-11 Recently, a number of research groups have used MALDI-TOFMS to identify and characterize bacteria and viruses rapidly.12-19 The application of this powerful tool to characterize fungal cells, * To whom correspondence should be addressed. Phone: 301-405-8617. Fax: 301-405-8615. E-mail:
[email protected]. (1) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (3) Montaudo, M. S.; Puglisi, C.; Samperi, F.; Montaudo, G. Rapid Commun. Mass Spectrom. 1998, 12, 519-528. (4) Westman, A.; Nilsson, C. L.; Ekman, R. Rapid Commun. Mass Spectrom. 1998, 12, 1092-1098. (5) Easterling, M. L.; Colangelo, C. M.; Scott, R. A.; Amster, I. J. Anal. Chem. 1998, 70, 2704-2709. (6) Uttenweiler-Joseph, S.; Moniatte, M.; Lambert, J.; Van Dorsselaer, A.; Bulet, P. Anal. Biochem. 1997, 247, 366-375. (7) Danis, P. O.; Karr, D. E. Rapid Commun. Mass Spectrom. 1996, 10, 862868. (8) Foret, F.; Mu ¨ ller, O.; Thorne, J.; Go¨tzinger, W.; Karger, B. L. J. Chromatogr. A 1995, 716, 157-166. (9) Montaudo, G.; Montaudo, M. S.; Puglisi, C.; Samperi, F. Rapid Commun. Mass Spectrom. 1995, 9, 453-460. (10) Yu, W.; Vath, J. E.; Huberty, M. C.; Martin, S. A. Anal. Chem. 1993, 65, 3015-3023. (11) Huberty, M.; Vath, J. E.; Yu, W.; Martin, S. A. Anal. Chem. 1993, 65, 27912800. (12) Holland, R. D.; Wilkes, J. G.; Rafii, F.; Sutherland, J. B.; Persons, C. C.; Voorhees, K. J.; Lay, J. O., Jr. Rapid Commun. Mass Spectrom. 1996, 10, 1227-1232. (13) Krishnamurthy, T.; Ross, P. L. Rapid Commun. Mass Spectrom. 1996, 10, 1992-1996. (14) Arnold, R. J.; Reilly, J. P. Rapid Commun. Mass Spectrom. 1998, 12, 630636.
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however, has received less attention.20,21 Rapid identification of fungi is of interest in medicine, in which accurate and timely identification of infectious agents plays an important role in effective treatment of disease. Identification of fungi is also of interest in food science and food production technologies. The kingdom Fungi includes a wide variety of members that range from simple unicellular species to more complex organisms. Diseases caused by fungi in plants and animals often require extensive treatment and are not normally responsive to traditional drug therapies. Of course, not all fungi are pathogenic. For example, archeological findings point to the fact that Saccharomyces cerevisiae was first used in wine making as early as 6000 BC in Mesopotamia (i.e., the present-day home of the Kurds) and in Egypt and Phoenicia around 5000 BC.22 The same organism is also known as “baker’s yeast” for its important role in bread making. Through an international effort, the genome of S. cerevisiae was the first eukaroytic genome to be completely sequenced and reported.23 The 12,068 Kb genome contains 5885 protein-encoding genes.23 This makes S. cerevisiae an excellent candidate to study by mass spectrometry and bioinformatics. Phyloproteomics is an emerging field in which organisms are identified by means of the masses of their proteins. In this approach, molecular weights of a microorganism’s proteins, obtained from its mass spectrum, are compared to those in genome and protein databases. Efforts are underway to develop software to carry out these searches and to weight the reliability of matches and species characterizations based on protein mass matches.24-26 (15) Hathout, Y.; Demirev, P. A.; Ho, Y. P.; Bundy, J. L.; Ryzhov, V.; Sapp, L.; Stutler, J.; Jackman, J.; Fenselau, C. Appl. Environ. Microbiol. 1999, 65, 4313-4319. (16) Cain, T. C.; Lubman, D. M.; Weber, W. J., Jr. Rapid Commun. Mass Spectrom. 1994, 8, 1026-1030. (17) Leenders, F.; Torsten, H. S.; Kablitz, B.; Franke, P.; Vater, J. Rapid Commun. Mass Spectrom. 1999, 13, 943-949. (18) Wang, Z.; Russon, L.; Li, L.; Roser, D.; Long, S. R. Rapid Commun. Mass Spectrom. 1998, 12, 456-464. (19) Dai, Y.; Li, L.; Roser, D.; Long, S. R. Rapid Commun. Mass Spectrom. 1999, 13, 73-78. (20) Welham, K. J.; Domin, M. A.; Johnson, K.; Jones, L.; Ashton, D. S. Rapid Commun. Mass Spectrom 2000, 14, 307-310. (21) Li, T.-Y.; Liu, B.-H.; Chen, Y.-C. Rapid Commun. Mass Spectrom. 2000, 14, 2393-2400. (22) Pretorius, I. S. Yeast 2000, 16, 675-729. (23) Goffeau, A.; Barrell, B. G.; Bussey, H.; Davis, R. W.; Dujon, B.; Feldmann, H.; Hoheisel, J. D.; Jacq, C.; Johnston, M.; Mewes, E. J. L.; Murakakmi, Y.; Philippsen, P.; Tettelin, H.; Oliver, S. G. Science 1996, 274, 546-567. (24) Demirev, P. A.; Ho, Y. P.; Ryzhov, V.; Fenselau, C. Anal. Chem. 1999, 71, 2732-2738. 10.1021/ac010651t CCC: $20.00
© 2001 American Chemical Society Published on Web 10/04/2001
EXPERIMENTAL SECTION Microorganisms. Candida albicans (ATCC 14053), Epidermophyton floccosum (ATCC 9646), and Saccharomyces cerevisiae (ATCC 26108) were purchased from the American Type Culture Collection (Rockville, MD). These microorganisms were grown in our laboratory according to the ATCC recommended procedures. The microorganisms were grown on agar composed of 21 g/L of YM broth and 6 g/L of bacto agar for S. cerevisiae and C. albicans and 39 g/L potato dextrose agar for E. floccosum. The materials for the growth media were purchased from Becton Dickinson and Company (Sparks, MD). After the required time for growth (24-72 h), the cultures were collected and suspended in sterile broth (21 g/L of YM broth for all organisms). These suspensions were then shaken for 24-72 h in the incubator. The final suspensions were then centrifuged at 8000g for 10-20 min. The pellets were washed three times with deionized/distilled sterile water. Following the washes, the cells were lyophilized and stored at -20 °C for future analyses by mass spectrometry. Cell Wall Lyses Techniques. A number of different strategies were investigated for rapid cell wall lysis. The methods that were used included exposing the intact cells to high concentrations of acids, such as trifluoroacetic acid, formic acid, nitric acid, and acetic acid. To prevent protein degradation due to the acids, the length of the exposure was kept to 5 min. Other methods evaluated included enzyme cleavage (Zymolyase purchased from Seikagaku American, Inc., Ijamsville, MD), direct protein extraction (Y-PER-R Yeast Protein Extraction Reagent, purchased from Pierce, Rockford, IL), ultrasonication, glass beads, and corona plasma discharge. Mass Spectrometry Analysis. A Kratos MALDI 4 TOF mass spectrometer (Kratos Analytical Instruments, Chestnut Ridge, New York) was employed. This mass spectrometer is equipped with an ultraviolet laser emitting at 337 nm with adjustable laser power. The fungal cells were suspended in a 70:30 (v/v) solution of acetonitrile:0.1% trifluroroacetic acid. The final concentration of the fungal cells was 5.0 mg/mL. A 0.2-µL aliquot of the sample solution was deposited on the sample slide and allowed to dry at room temperature. Each sample spot was covered with 0.2 µL of a 25% formic acid solution and allowed to stand for 5.0 min before it was dried under gentle airflow. In the final stage of sample preparation, a 0.2 µL portion of the matrix solution (usually a 50 mM solution of sinapinic acid) was deposited on each of the dried sample spots. The matrixes that were used included 2,5-dihydroxybenzoic acid (DHB), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), R-cyano-4-hydroxycinnamic acid (HCCA), 2,4,6-trihydroxyacetophenone (THAP), 4-hydroxy-3-methoxycinnamic acid (ferulic acid), and 3-hydroxypicolinic acid (HPA). After the matrix dried at room temperature, the samples were analyzed using the UV-MALDI-TOF mass spectrometer. Mass assignments were based on an external calibration spectrum, and endogenous ubiquitin was used as an internal standard. Protein Database Searches. To tentatively identify the protein peaks from the S. cerevisiae spectra, database searches were conducted using the Sequence Retrieval System (SRS) module (at http://expasy.hcuge.ch/srs5/) in the SwissProt/ (25) Pineda, F. J.; Lin, J. S.; Fenselau, C.; Demirev, P. A. Anal. Chem. 2000, 72, 3739-3744. (26) Demirev, P. A.; Lin, J. S.; Pineda, F. J.; Fenselau, C. Anal. Chem. 2001, 73, 4566-4573.
Figure 1. Matrix-assisted laser-desorption/ionization mass spectrum of C. albicans cells that were lysed by 25% formic acid. Sinapinic acid was used as the matrix.
Figure 2. Matrix-assisted laser-desorption/ionization mass spectrum of S. cerevisiae cells that were lysed by 25% formic acid. Sinapinic acid was used as the matrix.
TrEMBL (Expasy, the Swiss Institute of Bioinformatics, Geneva, Switzerland). In such searches, the average masses of the peaks from the spectra are entered into an interactive window (Alternative Query Form option in the menu). The only restrictions provided to the database search program were the masses of the unknown proteins and the name of the organism (i.e., protein subset from S. cerevisiae). A window of (3 Da was used. RESULTS AND DISCUSSION Microorganism Spectra. Three fungi were studied in the preliminary investigation of the application of UV-MALDI-TOF for the characterization of fungi. These microorganisms were C. albicans (ATCC 14053), S. cerevisiae (ATCC 26108), and E. floccosum (ATCC 9646). All three microorganisms showed very distinctive spectra, which could be used to distinguish them readily. The spectra of S. cerevisiae and C. albicans are depicted in Figures 1 and 2, respectively, for illustration. As can be seen in these figures, the spectrum of S. cerevisiae exhibits more peaks in the higher mass region (i.e., above 4000 Da). Cell Wall Lyses Techniques. Several techniques were investigated for lysis of the cell wall of S. cerevisiae. The methods Analytical Chemistry, Vol. 73, No. 21, November 1, 2001
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Table 1. Tentative Identification and Characterization of Proteinsa Desorbed from S. cerevisiae MH+ 3301 5856 5942 5988 6019 6212 6532 6597 6741 8558 a
database match (MH+)
entry name
theoretical pI
GRAVY
identity
3299 5853 5941 5986 6016 6211 6530 6531 6531 6597 6740 8558
Q36739 Q96109 Q9ZZW3 OM05_YEAST RL40_YEAST RL39_YEAST R29A_YEAST Q05444 Q05441 R29B_YEAST OM07_YEAST UBIQ_YEAST
9.53 9.99 6.81 8.11 10.32 12.55 10.31 5.26 4.72 10.07 8.22 6.56
0.524 1.292 0.134 -0.640 -0.990 -1.494 -0.898 -0.249 -0.256 -0.955 0.183 -0.513
cytochrome c oxidase polypeptide I (encoded in mitochondria) mitochondrion OXI3 ORF Q0143 (encoded in mitochondria) mitochondrial import receptor subunit TOM5 60S ribosomal protein L40 60S ribosomal protein L39 40S ribosomal protein S29-A subunit IV cytochrome c oxidase [precursor] [fragment] subunit IV cytochrome c oxidase [precursor] [fragment] 40S ribosomal protein S29-B mitochondrial import receptor subunit TOM7 ubiquitin
Proteins from Figure 2.
Figure 3. Matrix-assisted laser-desorption/ionization mass spectrum of S. cerevisiae cells that were lysed by corona plasma discharge. Sinapinic acid was used as the matrix.
included exposing the intact cells to high concentrations of acids, Zymolyase, protein extraction reagent, ultrasonication, glass beads, and corona plasma discharge. Figures 2 and 3 depict spectra obtained using two different methods, a high concentration of formic acid, and corona plasma discharge, respectively. The best spectra for S. cerevisiae were obtained when the intact cells were exposed to a 25% formic acid solution for 5 min immediately before mass spectrometric analysis. The qualities of the spectra obtained using the other cell wall lyses techniques listed in the Experimental Section, in terms of number of peaks and signal-to-noise ratio, fall between the two examples shown. The choice of cell wall lysis technique was seen to play a very important role in determining the number of proteins desorbed, as well as the signal-to-noise ratio of the spectra obtained, as is apparent in Figures 2 and 3. A list of the protein peaks desorbed in the MALDI spectrum of S. cerevisiae is reported in Table 1. Matrix Evaluation. A number of MALDI matrixes were evaluated; they included DHB, sinapinic acid, and R-cyano-4hydroxycinnamic acid. It was expected that different matrixes would produce different results; however, this evaluation was carried out to find the matrix that provided the most peaks in the mass range 4-20 K Da. Dihydroxybenzoic acid (DHB) produced the poorest spectra; on the other hand, the use of sinapinic acid resulted in spectra with much higher quality (i.e., number of peaks 5230 Analytical Chemistry, Vol. 73, No. 21, November 1, 2001
Figure 4. Matrix-assisted laser-desorption/ionization mass spectrum of S. cerevisiae cells that were lysed by 25% formic acid. Dihydroxybenzoic acid was used as the matrix.
and signal-to-noise ratio). This is illustrated by comparing Figures 2 and 4, in which sinapinic acid and dihydroxybenzoic acid were used as the matrix, respectively. The qualities of the spectra obtained using the other matrixes listed in the Experimental Section, in terms of number of peaks and signal-to-noise ratio, fall between the two examples shown. On the basis of the results obtained in this part of the preliminary investigation, sinapinic acid (50 mM) was chosen as the matrix of choice to study S. cerevisiae. Characterization of Protein Biomarkers. Bioinformatics software was used to match the masses observed to protein masses generated in silico from protein and genome databases. Among the peaks in Figure 2, matches were found and identities are proposed for 10, those shown in Table 1. Immediately of note, protonated ubiquitin is identified at m/z 8558. Since its discovery in 1975,27 this protein has been found to be abundant in eukaroytes and absent in prokaryotes. Its mass varies across the kingdom (between 8500 and 8600 Da), for which it can serve as a biomarker. Furthermore, the mass differences can be used as a means to distinguish different organisms within this kingdom. Most of the proteins tentatively identified in Table 1 are basic, including four candidates from the ribosome and five from the (27) Goldstein, G.; Scheid, M.; Hamerling, U.; Boyse, E. A.; Schlesinger, D. H.; Niall, D. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 1-15.
mitochondria. The detection of ribosomal proteins is not unexpected, because ribosomes constitute a large portion of the mass of a growing cell, and most of their proteins are strongly basic.28,29 Putative observation of mitochondrial proteins constitutes a major difference in the MALDI spectra of fungi, as compared with prokaryotes, which lack this organelle. The observation of biomarkers from cytosolic organelles (absent in Figure 3) indicates the cell wall is effectively lysed during sample preparation with 25% formic acid. Another 10 or so prominent peaks in Figure 2 could not be identified by direct matching of observed and theoretical masses. Most of these fall below m/z 4000 and are probably not genomically translated proteins.24 Unidentified peaks in the mass range above 4000 may be posttranslationally modified. Characterization of these biomarkers is being pursued. Hydropathicity of the Proteins. The grand average of hydropathicity (listed as GRAVY in Table 1 as used by Expasy, the Swiss Institute of Bioinformatics, Geneva, Switzerland), is the measure of how hydrophilic or hydrophobic a protein is. The more hydrophilic a protein, the more negative its GRAVY value would be; and the more hydrophobic, the more positive its GRAVY value. (28) Arnold, R. J.; Reilly, J. P. Anal. Biochem. 1999, 269, 105-112. (29) Ryzhov, V.; Fenselau, C. Anal. Chem. 2001, 73, 746-750. (30) Todd, P. J.; Schaaff, T. G.; Chaurand, P.; Caprioli, R. M. J. Mass Spectrum. 2001, 36, 355-369. (31) Rubakhin, S. S.; Garden, R. W.; Fuller, R. R.; Sweedler, J. V. Nature Biotechnol. 2000, 18, 172-175.
These theoretical values are based on primary sequence and do not reflect the changes brought about by protein folding and other structural changes; hence, they should be looked at with some caution. The majority of the proteins desorbed are hydrophilic, reflecting the conditions provided by the solvents used to obtain the MALDI spectra. CONCLUSIONS This report provides evidence that many of the biomarkers desorbed from eukaroytic S. cerevisiae are unmodified proteins and can be tentatively characterized by mass-based database searching. These observations support not only the ongoing development of MALDI mass spectrometry for rapid characterization of fungi, but also its extension to analyses of human and other eukaroytic tissue.30,31 ACKNOWLEDGMENT The authors thank Dr. P. Demirev for useful discussions throughout the course of the project and Ms. A. Freas for her work in growing the microorganisms for this project. Funding for this project was provided by Defense Advanced Research Project Agency and National Science Foundation. Received for review June 12, 2001. Accepted August 8, 2001. AC010651T
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