Anal. Chem. 1999, 71, 1990-1996
Monitoring the Growth of a Bacteria Culture by MALDI-MS of Whole Cells Randy J. Arnold, Jonathan A. Karty, Andrew D. Ellington,† and James P. Reilly*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
We have probed the time evolution of a growing bacteria culture by extracting samples periodically and performing matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) on whole cells. The mass spectra generated by this method contain tens of peaks in the 3-11-kDa mass range. Cultures of E. coli strain K-12 were grown in two types of containers and at two nutrient concentrations and sampled periodically from 6 to 84 h after inoculation. The relative intensities of several of the stronger peaks vary quite dramatically as a function of time. These temporal characteristics must be taken into account when MALDI-MS is applied to identify bacteria. The results also suggest that MALDI-MS can be used to follow the aging of a bacteria culture. Mass spectrometry has been under investigation as a method for characterizing bacteria since the mid-1970s.1 Pyrolysis mass spectrometry,2-4 gas chromatrography/mass spectrometry, 5 and fast-atom bombardment mass spectrometry6-8 have all been employeed. Recently, a number of reports have described the use of matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry for bacterial identification. The feasibility of generating MALDI mass spectra from bacterial cell extracts,9 the effect that sample preparation technique has on the reproducibility of MALDI mass spectra,10,11 and the variations among different species and strains12-16 have all been investigated. In † Present address: Department of Chemistry, Institute of Cellular and Molecular Biology, A4800, University of Texas Austin, Austin, TX 78712. (1) Anhalt, J. P.; Fenselau, C. Anal. Chem. 1975, 47, 219-225. (2) DeLuca, S.; Sarver, E. W.; Harrington, P. d. B.; Voorhees, K. J. Anal. Chem. 1990, 62, 1465-1472. (3) Snyder, A. P.; Smith, P. B. W.; Dworzanski, J. P.; Meuzelaar, H. L. C. ACS Symp. Ser. 1994, No. 541, 62. (4) Basile, F.; Voorhees, K. J.; Hadfield, T. L. Appl. Environ. Microbiol. 1995, 61, 1534-1539. (5) Fox, A.; Rosario, R. M. T.; Larsson, L. Appl. Environ. Microbiol. 1993, 59, 4354-4360. (6) Heller, D. N.; Murphy, C. M.; Cotter, R. J.; Fenselau, C.; Uy, O. M. Anal. Chem. 1988, 60, 1787-2791. (7) Cole, M. J.; Enke, C. G. Anal. Chem. 1991, 63, 1032-1038. (8) Heller, D. N.; Cotter, R. J.; Fenselau, C.; Uy, O. M. Anal. Chem. 1987, 59, 2806-2809. (9) Cain, T. C.; Lubman, D. M.; Weber, W. J., Jr. Rapid Commun. Mass Spectrom. 1994, 8, 1026-1030. (10) Liang, X.; Zheng, K.; Qian, M. G.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1996, 10, 1219-1226. (11) Wang, Z.; Russon, L.; Li, L.; Roser, D. C.; Long, S. R. Rapid Commun. Mass Spectrom. 1998, 12 456-464.
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some of these studies, mass spectra of unknown strains are compared to libraries of mass spectra from known bacteria. The identity of the unknown is then discerned by searching for distinctive features in the mass spectrum12-15 or by mathematically comparing the spectra.16 Unfortunately, these methods are often hampered by the poor reproducibility of bacterial mass spectra.11 Despite rigorous controls on sample handling and mass spectrometric conditions, spectra obtained from two different samples of the same strain can vary to some degree.11 Some of this variation in peak intensities can be attributed to the MALDI process. However, biological factors also play a role. Holland and co-workers reported that the mass spectrum of a stored colony of bacteria changed when the same colony was sampled over a period of several days.17 This has serious implications if the mass spectrum of an unknown strain is to be compared to a library. Establishing a match between two bacteria sample strains may require that the cultures from which their spectra were recorded be of similar age. Bacteria adapt very rapidly to their environment. A flask of bacterial growth medium is a relatively closed system. Although fresh air can enter and leave aerobically grown cultures, limited essential nutrients in the medium are utilized, and the bacteria produce metabolic products that modify the chemical makeup of the medium.18 The organisms, in turn, adapt to their changing chemical environment, and this may affect their mass spectra. In this study, the mass spectra of bacterial colonies were recorded at regular intervals for a period of up to 84 h. Culture volume and initial nutrient concentration were also varied in order to determine how these parameters affect the time dependence of the mass spectra. (12) Holland, R. D.; Wilkes, J. G.; Sutherland, J. B.; Persons, C. C.; Voorhees, K. J.; Lay, J. O., Jr. Rapid Commun. Mass Spectrom. 1996, 10, 1227-1232. (13) Claydon, M. A.; Darey, S. N.; Edwards-Jones, V.; Gordon, D. B. Nature Biotechnol. 1996, 14, 1584-1586. (14) Krishanmurthy, T. Ross, P. L. Rapid Commun. Mass Spectrom. 1996, 10 1992-1996. (15) Krishnamurthy, T.; Ross, P. L.; Rajamani, U. Rapid Commun. Mass Spectrom. 1996, 10, 883-888. (16) Arnold, R. J.; Reilly, J. P. Rapid Commun. Mass Spectrom. 1998, 12, 630636. (17) Holland, R. D.; Burns, G.; Persons, C. C.; Rafii, F.; Sutherland, J. B.; Lay, J. O., Jr. Proceedings from the 45th American Society for Mass Spectrometry Conference on Mass Spectrometry and Related Topics, Palm Springs, CA, 1997; p 1353. (18) Gerhardt, P.; Drew, S. P. In Methods for General and Molecular Bacteriology; Gerhardt, P., Murray, R. G. E., Wood, W. A., Krieg, N. R., Eds.; ASM Press: Washington, DC, 1994. 10.1021/ac981196c CCC: $18.00
© 1999 American Chemical Society Published on Web 04/09/1999
Table 1. Culture Growth Conditions culture
culture vol (mL)
flask vol (mL)
medium
A B C
200 5 200
1000 25 1000
LB LB 10% LB in water
EXPERIMENTAL SECTION Three different sets of growth conditions were examined in this study. Culture A was grown in a 1000-mL Erlenmeyer flask filled with 200 mL of Luria Bertani (LB) medium.19 Culture B was grown in a set of 30 replicate 16 × 125 mm culture tubes each containing 5 mL of LB medium. Culture C was grown in a 1000mL Erlenmeyer flask filled with 200 mL of a solution consisting of 9 parts distilled water and 1 part LB medium. These conditions are summarized in Table 1. All cultures used in this study were grown in house at 37 °C shaking at 200 rpm. They were inoculated from a starter culture of K-12 Escherichia coli (ATCC 25404, obtained from the Indiana University Biology Department) that had been grown the previous night. Cultures A and C were inoculated with 40 µL of this starter culture, and each test tube of culture B was inoculated with 1 µL of the starter culture in order to maintain the same volume ratio of inoculum to culture. Duplicate 1-mL aliquots were sampled from culture A and single 1-mL aliquots were sampled from cultures B and C at 2-h intervals from 6 to 48 h after inoculation. Cells were harvested by centrifugation (7000g for 5 min) and washed once with a solution of 50 mM glucose and 10 mM EDTA in order to inhibit their growth and keep them intact. Cells were centrifuged again, and the resulting pellet was stored dry at -20 °C for at least 12 h before analysis. At harvest, the optical density (OD) at 600 nm was measured for each culture against a blank of the respective growth medium using a Hitachi 300 spectrophotometer. Samples with an OD greater than 1 were diluted with either 1 or 9 parts growth medium to reduce the absorbance and improve the accuracy of its measurement. The reported OD values have had this dilution taken into account. To ensure that approximately the same number of cells were in each MALDI spot, the cell pellets were diluted with 40 µL of distilled water per unit of OD. MALDI spots were made by mixing 1 µL of bacterial suspension with 9 µL of a 10 mg/mL solution of R-cyano-4-hydroxycinnamic acid (CHCA, Aldrich Chemical) dissolved in 2 parts 0.1% trifluoroacetic acid (TFA, obtained from Fisher Scientific) and 1 part acetonitrile (Fisher Scientific). One microliter of this solution was deposited on a stainless steel probe and allowed to air-dry. Mass spectra were recorded using a previously described home-built time-of-flight mass spectrometer.20 MALDI spots were irradiated with 355-nm light from a frequency-tripled Nd:YAG laser. Positive ions were accelerated through 20 kV toward a dual microchannel plate detector. Spectra were recorded on a HewlettPackard model 54270D digital oscilloscope at a sampling rate of 500 MHz and signal averaged for 200 or 500 shots. (19) Miller, J. H. Experiments in Molecular Genetics; Cold Spring Harbor Press: Cold Spring Harbor, NY, 1972. (20) Christian, N. P.; Colby, S. M.; Giver, L.; Houston, C. T.; Arnold, R. J.; Ellington, A. D.; Reilly, J. P. Rapid Commun. Mass Spectrom. 1995, 9 10611066.
After acquisition, data were processed with a fast Fourier transform (FFT) algorithm to remove baseline drift and some of the high-frequency noise.16 Doubly and singly charged ions of lysozyme, cytochrome c, and ubiquitin (all obtained from Sigma Chemical) were used as external mass calibrants, and the TOF data were fit to the equation m/z ) A(TOF)2 + B(TOF) + C + D(TOF)1/2 and plotted on a linear mass scale. Spectra were compared using a previously described modified cross-correlation algorithm.16 To assist in the identificaton of some of our mass spectral peaks, liquid-phase isoelectric focusing (IEF) experiments were carried out on proteins extracted from E. coli strain K-12 cells. Cells were grown for 21.5 h in normal LB media and harvested by centrifugation. The resulting pellet was diluted with distilled water and centrifuged at 10000g for 10 min. The supernatant was decanted and retained. Biolyte pH 3-10 ampholyte (40%, Bio-Rad) was added to the supernatant so that the ampholyte concentration in the sample was 0.01%. The Rotofor IEF cell (Bio-Rad) was filled with 50 mL of this solution. The anode and cathode chambers were filled with 0.1 M NaOH and 0.1 M H3PO4, respectively. A constant 12 W of power was supplied to the cell for 3 h after which the current dropped from 34 to 8 mA and remained constant, indicating that proteins were focused. Twenty fractions were collected, and the pH of each was measured. Their values ranged from 1.51 to 12.32 pH units. RESULTS Spectral Variation. Samples of culture A were extracted every 2 h from 6 to 48 h after inoculation and at selected times past 48 h. Mass spectra of these samples (series A) are shown in Figure 1. This figure illustrates qualitative differences in the spectra that depend on culture growth time. Previously we demonstrated that when bacteria cultures are grown and handled using the same protocol, virtually identical mass spectra can be recorded.16 Thus, the large variations displayed by the spectra in Figure 1 are very significant. The relative lack of high-mass (greater than ∼8 kDa) peaks early in culture growth, the variations in peak intensities with growth time, and the delayed appearance of certain peaks are trends that we previously observed in growth studies on other E. coli strains (data not shown). Peaks at 3637, 4364, 5096, 6254, 7274, 7333, and 8326 Da dominate the spectra at early growth times, and their intensities fade as the culture ages. The 7274and 3637-Da features appear to be the singly and doubly charged ions of the same protein. Five of the peaks, including 3637 and 7274 Da, correspond to four ribosomal proteins, as will be discussed later. The 7333- and 8326-Da ions have not been conclusively identified but tentative assignments will be presented below. Peaks at 4939, 5878, 6100, 7149, 8447, 9877, and 10 314 Da do not appear until several hours after inoculation. An interesting array of evenly spaced peaks at the low-mass end of our spectra emerge at ∼40 h are present through 48 h and then disappear ∼60 h after inoculation. These peaks are separated by 129.2 ( 0.2 Da, but we currently have no other information relating to their identity. Spectra were also recorded for a duplicate set of culture A samples that were handled separately (data not shown). The basic trends just described involving peaks at higher masses becoming more intense over time, specific peaks appearing and disappearing during culture growth, and evenly spaced peaks at lower mass emerging at longer growth times were clearly Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
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Figure 1. Mass spectra of E. coli strain K-12 grown in 200 mL of LB growth media (series A), sampled at various times after inoculation.
observed. Except for the evenly spaced low-mass peaks, these features were also observed in series B spectra obtained from culture B (grown in 5-mL test tubes). Series C spectra obtained from culture C (grown in dilute growth medium) displayed all the trends mentioned here for series A, although intensities of some peaks show significant differences from one culture to another and the time scales of their variations differ in some cases. A thorough study of these differences will become more useful when the identities of these peaks are determined. Growth Phases. The progression of the culture through its growth cycle may be responsible for much of the observed spectral variation. As the medium in which cells are growing becomes depleted in nutrients and saturated with waste products, the cells must adapt. The phases of culture growth, lag (initial adjustment), log(exponential growth in plentiful nutrients), stationary (nutrient limit met, growth ceases), and death (waste accumulates, cells die),21 are commonly monitored by measuring the turbidity or OD of the culture. Figure 2 shows a plot of OD vs time for culture A samples. From this figure it is clear that the 1992 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
culture has experienced the lag, log, and stationary phases and entered the death phase in the course of this work. Peak Identification. Reacting our samples with trypsin eliminates all peaks in the mass spectrum. We conclude that the majority of the observed peaks are due to cellular proteins, consistent with previous suggestions.9,12,15 The intensities of mass spectral peaks presumably reflect molecular concentrations in the cells. Because proteins vary in their MALDI ionization efficiencies, especially when they are components of mixtures, the MALDI process is not optimal for quantitative comparison of different substances. This is due to a combination of factors, including protein solubility, presence of ionizable functional groups, tertiary structure, and ability of the protein to incorporate into the matrix crystal. However, since relative peak intensities for different molecules in bacteria mass spectra can be reproducible,16 it should be possible to learn about how individual proteins vary with time by comparing spectra. Identification of some of the more dominant (21) Neidhardt, F. C.; Ingraham, J. L.; Schaechter, M. Physiology of the Bacterial Cell; Sinauer Assoc., Inc.: Sunderland, MA, 1990; Chapters 7 and 15.
Figure 2. Optical density (600 nm) of E. coli strain K-12 grown in 200 mL of LB growth media (series A) plotted vs growth time.
Figure 3. Mass spectra of (A) ribosomes extracted from E. coli strain K12 cells grown for 12.5 h, and series A spectra of whole cells grown for (B) 12, (C) 20, and (D) 30 h.
peaks in our spectra should provide further insight into the protein content of cells during culture growth. Peaks at 4364, 5096, 5380, 6254, 6315, 6411, and 7274 Da that appear throughout the growth study spectra are also observed in the MALDI mass spectra of whole ribosomes extracted from an ∼12.5-h-old culture of E. coli strain K-12.22 These masses correspond to singly charged ions of ribosomal subunit proteins L36, S22, L34, L33, L32, L30, and L29, respectively. Figure 3A displays a mass spectrum of the ribosome sample and, for comparison, mass spectra of bacteria whole cells grown for 12, 20, and 30 h (22) Arnold, R. J.; Reilly, J. P. Anal. Biochem., in press.
Figure 4. Peak height ratios for ribosomal subunit proteins L36, S22, L34, L33, L32, L30, and L29 calculated by dividing the height of each peak by the height of the corresponding peak in the 10-h series A spectrum as a function of culture growth time. The solid curve displays the average of the seven points at each growth time.
(series A) appear in Figure 3B-D. The similarity of the 12-h whole cell spectra to the ribosomal spectrum reflects the abundance of ribosomal proteins in cells at this stage of growth. Peak height ratios were calculated by dividing the height of each ribosomal peak by the height of the corresponding peak in the 10-h series A spectrum. The seven peak height ratios for each spectrum are plotted as a function of culture growth time in Figure 4. The solid curve displays the average of the seven points at each growth time. The ribosomal protein abundance obviously decreases with increasing growth time. The result is consistent with our understanding that cells grow more rapidly at early times when nutrients are plentiful and they assemble ribosomes to produce proteins at the highest rate during this period.23,24 At later times, cells grow less rapidly and fewer ribosomes are required for protein synthesis. The data are also consistent with the fact that these ribosomal proteins are present in equal numbers so all masses follow the same basic trend. The proteins of E. coli and several other organisms are tabulated in a number of databases on the Internet. These databases contain information such as amino acid sequence, mass, pI, and structural data. Using a mass-specific search engine to access these databases, it is possible to find tentative assignments for peaks in our spectra.25-27 However, a variety of posttranslational modifications, including methylation, acetylation, carboxylation, and protease cleavage, may occur for the proteins we observe.28 In searching for tentative assignments, we considered only the masses of unmodified proteins and of proteins that had lost an N-terminal methionine. The peak at 7333 Da can be matched to either cold shocklike protein CSPE (7332.3 Da) or entry exclusion (23) Wittmann, H. G. Am. Rev. Biochem. 1982, 51, 155. (24) Bremer, H.; Dennis, P. O. In Escherichia Coli and Samonella Neidhardt, F. D., Ed.; ASM Press: Washington, DC, 1996; p 167. (25) Peter, J.; Quadroni, M.; Carafoli, E.; Gonnet, G. Biochem. Biophys. Res. Commun. 1993, 195, 58-64. (26) Pappin, D. J. C.; Hojrup, P.; Bleasby, A. J. Curr. Biol. 1993, 3, 327-332. (27) Mann, M.; Hojrup, P.; Roepstorff, P. Biol. Mass Spectrom. 1993, 22, 338332. (28) Krishna, R. G.; Wold, F. In Advances in Enzymology and Related Areas of Molecular Biology; Meister, A., Ed.; John Wiley and Sons: New York, 1993; Vol. 67, pp 265-296.
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Table 2. Possible Peak Assignments obsd mass
obsd pI range
protein name
sequence massa (Da)
sequence pIa
7333
8.7-9.2
8326
4.6-5.9
cold shocklike protein CSPE entry exclusion major outer membrane lipoprotein precursor 8.3-kDa protein in DINF-QOR intergenic reg. dihydrofolate reductase type II
7332.3 7333.6 8323.5 8325.3 8328.4
8.06 8.88 9.30 5.44 6.81
a Sequence mass and pI returned from MS-Fit search of Swiss Prot and Genpept databases. Ms-Fit can be accessed at http://prospector. ucsf.edu/mshome.htm.
protein B (7333.6 Da). The peak at 8326 Da may correspond to either major outer membrane lipoprotein precursor (8323.5 Da), 8.3-kDa protein in DINF-QOR intergenic region (8325.3 Da), or dihydrofolate reductase type II (8328.4 Da). In these cases, our mass accuracy does not allow us to eliminate any of these possible matches. However, liquid-phase isoelectric focusing of the supernatant recovered from E. coli whole cell extracts provides additional information. MALDI mass spectra of IEF fractions indicate that the pI of the protein associated with the 7333-Da peak is between approximately 8.7 and 9.2. The pI for the protein corresponding to the peak at 8326-Da peak is between approximately 4.5 and 5.7. These results support the assignment of the 7333-Da peak as entry exclusion protein B (pI of 8.88) and the 8326-Da peak as the 8.3-kDa protein in DINF-QOR intergenic region (pI of 5.44). The data are summarized in Table 2. The peaks that appear late in the growth study are not tentatively assigned since IEF data have not yet been obtained for these proteins and the database searching did not lead to obvious matches for most of those masses. The identification of these and other spectral features is currently being pursued. Correlation Analysis. Figure 1 displays a significant amount of spectral variation with culture growth time. This variation provides a challenge when using mass spectrometry for bacteria identification, as one must take precautions to use sample cultures at about the same state of growth. This “same state” might not always mean the same time because other parameters such as temperature and the availability of nutrients and metabolites can also affect growth rate.21 Obviously, growth time is an important cause of spectral variation. The following results suggest that such variation can be monitored quantitatively. Cross-correlation analysis, as previously described,16 can be used to compare any two bacterial mass spectra. Whereas standard cross-correlation uses the entire spectral range to compute a single correlation value, in our modified cross-correlation approach, we divide each spectrum into a number of smaller intervals, and correlation coefficients associated with each of these smaller intervals are generated. These numbers are then multiplied to produce a composite correlation index that reflects the similarity of two spectra. This modified approach increases the sensitivity of correlation analysis to small spectral differences. The technique can be fine-tuned by varying the number of intervals into which the spectrum is divided. Previous work16 compared samples of different bacteria strains that were grown for the same amount of time. Here we use the technique to quantitatively measure the degree to which spectra of the same strain change with growth time. For these comparisons, we divided the mass range from 3500 to 10 000 Da into 13 intervals of 500 Da each. 1994 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
Figure 5. Composite correlation index values for growth series A spectra compared to 10- (O), 20- (4), 30- (0), and 40- (3) h series A spectra, plotted on (A) linear and (B) logarithmic scales.
Figure 5 shows the effect of using this cross-correlation approach to compare samples grown in 200 mL of LB medium (culture A) for four particular lengths of time (10, 20, 30, and 40 h) with all series A cell cultures sampled from 6 to 48 h. Notice that, for each of the four curves, nearby time points compare favorably, but the composite correlation index decreases as the time difference increases. Part A of the figure is plotted on a linear scale, illustrating that the composite correlation indexes are rather sharply peaked. Part B is plotted on a logarithmic scale and shows that as the growth times associated with two samples diverge, their composite correlation indexes monotonically decay over several orders of magnitude. This effect is not unique to these time points; spectra associated with other growth periods would yield curves similar to those in Figure 5. One question that arises is whether spectra change more dramatically at some times than at others. Figure 6 shows correlation index values calculated by comparing consecutive series A time points. Low values indicate that relatively dramatic changes are occurring while large values denote little or no change. This curve indicates that the spectra change more
Figure 6. Composite correlation index values for consecutive (2 h different) growth series A spectra.
Figure 8. Composite correlation index values for growth series B spectra compared to 10- (O), 20- (4), 30- (0), and 40- (3) h series A spectra, plotted on (A) linear and (B) logarithmic scales.
Figure 7. Mass spectra of E. coli strain K-12 grown in 5 mL of LB growth media (series B), sampled at selected times after inoculation.
dramatically at very early times (6-10 h) and from 22 to 30 h than at other times such as between 30 and 44 h. This result suggests that some periods of growth may be preferred to others for the purpose of comparing or identifying bacteria. The effects of varying the growth culture volume (5 mL of normal LB medium in test tubes, series B) and growth medium concentration (10% LB medium in a 200-mL flask, series C) on the bacterial mass spectra were also investigated. A few representative mass spectra from series B are displayed in Figure 7. Several familiar trends are observed. The mass spectrum of the 6-h sample has relatively few peaks, all at low masses. The 22-h mass spectrum looks very similar to earlier mass spectra with the addition of a prominent peak at 3076 Da that appears rather
suddenly. By 30 h the peak has disappeared almost entirely. This same peak is off-scale in the 24-h mass spectrum of series A. Just as in series A, higher mass peaks emerge as the colony ages. A peak at 8330 Da intensifies at 24 h and remains strong subsequently. Another interesting feature is the peak that appears at 8450 Da. At 24 h, it is barely perceptible, but by 34 h, it is easily visible. The 8450-Da peak seems to grow at the expense of the 8330-Da peak. The last three spectra in the figure show how the 8330-Da peak shrinks as the 8450-Da peak grows in intensity. The same trend is seen in the mass spectra of series A. It should also be noted that the ribosomal peaks at 4364, 5096, 6254, and 7273 Da dominate the early mass spectra and fade as the colony ages, in accord with the data from series A. Finally, as previously mentioned, the array of evenly spaced peaks seen in the later series A spectra did not reproduce in series B although it did occur in series C. Figure 8 shows composite correlation indexes for comparing 10-, 20-, 30-, and 40-h series A spectra to each 5-mL series B spectrum. These curves indicate that the smaller volume, test tube-grown cultures reach similar growth stages at slightly longer times than larger volume, flask-grown cultures. A similar comparison of the diluted growth medium spectra (series C) to series A spectra indicated that the cells may progress through the growth cycle slightly faster in the dilute medium (data not shown). However, neither growth medium concentration nor culture volume variations cause more significant spectral differences than are induced by large variations in culture growth time. DISCUSSION On the basis of the spectra shown in Figure 1, it is fair to conclude that MALDI mass spectra of whole bacteria cells change Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
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in a systematic way with growth time. Currently, we have a limited understanding of the factors responsible for the observed changes. The latter probably include the decreasing availability of nutrients and metabolites, increasing waste concentrations, cellular overcrowding, and increasing numbers of dead cells. Identification of the mass spectral peaks should facilitate the study of cell metabolism by this technique. Through our previous work on isolated ribosomes, we can assign seven features in these spectra to ribosomal proteins. This corresponds to a small fraction of the observed peaks, and identifying the others presents interesting challenges. An excellent approach for this involves deriving sequence information and matching it with predictions based on the previously determined E. coli genome.25-27 Because posttranslational modifications28 can change the mass of a protein from its predicted value, sequence information is particularly helpful in identifying proteins. Mass spectrometric techniques such as MALDI in-source, postsource, collisionally induced, and photo-fragmentation offer potential pathways to such sequence information.29-32 An alternative approach involves the use of enzymatic digestion followed by mass spectral analysis.33 These experiments are fairly straightforward to carry out for simple, onecomponent protein samples. However our spectra contain tens of peaks and since bacteria are known to produce over 2000 different proteins many heavy proteins are certainly present in our samples even though they do not appear in our mass spectra. This sample complexity has thus far prevented us from applying these techniques to obtain useful sequence information. However, in very recent work, Long, Li, and co-workers have demonstrated that HPLC separation of the bacterial proteins can solve this problem.34 These results have significant implications for the use of MALDI mass spectrometry in fingerprinting bacteria. As shown in Figure 1, some of the peaks vary from being dominant to being absent from the spectrum over a matter of hours. Identification techniques such as those based on individual peak matching14,17 can be problematic if the presence or absence of chosen biomarker peaks depends on growth time. The data displayed in Figure 6 for spectral comparison using the modified correlation technique suggest that at certain times during culture growth the spectra remain stable for a few hours and these times may be better suited for sampling than others. In general, special attention (29) Brown, R. S.; Lennon, J. J. Anal. Chem. 1995, 67, 3990-3999. (30) Kaufmann, R.; Spengler, B.; Lu ¨ tzenkirchen, F. Rapid Commun. Mass Spectrom. 1993, 7, 902-910. (31) Biemann, K. Biomed. Environ. Mass Spectrom. 1998, 16, 99-111. (32) Williams, E. R.; Furlong, J. J. P.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1990, 1, 288-294. (33) Scha¨r, M.; Bo ¨rnsen, K. O.; Gassmann, E. Rapid Commun. Mass Spectrom. 1991, 5, 319-336. (34) Dai, Y.; Li, L.; Roser, D. C.; Long, S. R. Rapid Commun. Mass Spectrom. 1999, 13, 73-78. (35) Gennis, R. B.; Stewart, V. Escherichia coli and Salmonella Cellular and Molecular Biology, 2nd ed.; ASM Press: Washington, DC, 1996; Vol. 1, Chapter 17.
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should be paid to ensure proper bacterial identification using mass spectrometry, including sampling the culture at a reproducible time or optical density. With the same concentration of growth medium, cells would be expected to grow at similar rates, although different volumes may allow other parameters such as oxygen concentration to vary. On the basis of composite correlation index values for comparing series B spectra to selected series A spectra (Figure 8), the smaller volume, test tube-grown culture B seems to progress through the growth cycle slower than the larger volume, flask-grown culture A. This slight deceleration may be due to a reduction in dissolved oxygen concentration in the test tubes compared to the large flask.35 In general, the composite correlation values for comparing these series of spectra are larger than those obtained by comparing 10% LB series C spectra to normal LB series A spectra, (data not shown), suggesting that changing the culture volume has a less detrimental effect on comparisons than diluting the growth medium by a factor of 10. In all cases, the greatest spectral differences are observed for large growth time differences, as shown in Figures 5 and 8. Future work will be aimed at identifying the proteins in our mass spectra. This should provide valuable insights into the cellular origin of these proteins and whether they are soluble in the cytoplasm, bound to membranes, or attached to the cell wall. Also, if these proteins are critical for cell growth, their identification would allow MALDI mass spectrometry to be used as a rapid method for monitoring cellular metabolism. CONCLUSIONS MALDI mass spectra of whole bacteria samples vary qualitatively and quantitatively with respect to culture growth time. Other parameters, such as growth medium volume and concentration, can also cause spectral changes, leading to poor correlation and possible misidentification. Spectral variation of the magnitude that we have observed can be detrimental to the utilization of mass spectrometry for bacterial identification. Some peaks that are strong at one time can be absent at other times during culture growth. Although this technique has been used to compare and identify bacterial strains, factors such as growth time must be controlled to ensure accurate results. ACKNOWLEDGMENT This work has been supported by the National Science Foundation. We thank Theodore Widlanski, Indiana University Department of Chemistry, for the use of his Rotofor Isoelectric Focusing Apparatus and Karl Kulow, Indiana University Department of Biology, for supplying the E. coli strain K-12 sample. Received for review November 3, 1998. Accepted February 24, 1999. AC981196C
