ORIGINAL PAPER

High-resolution MALDI-TOF mass spectrometry of bacterialproteins using a Tris-EDTA buffer approach

Faheem Ahmad & Hui-Fen Wu

Received: 26 May 2011 /Accepted: 9 September 2011 /Published online: 30 October 2011# Springer-Verlag 2011

Abstract The performance of matrix assisted laserdesorption-ionization time of flight mass spectrometry(MALDI-TOF) of bacterial proteins strongly depends onsample preparation. It is found that the mass spectralprofiles obtained from direct MALDI-TOF MS of theprotein extracts are much weaker for individual bacterialcells than compared to those prepared by the Tris-EDTAbuffer approach (TEBA). Characteristic mass spectral peakswere observed in the mass range from 3,000 to 15,000 Da.The mass peaks reported earlier and claimed to serve asspecies-specific biomarkers are consistently found hereas well. Mass peaks at m/z of 3636, 5466, 5750, 6315,6547, 7274, 9192, and 9742 are found for Escherichiacoli studied and assigned as specific biomarkers. Similar-ly, specific mass peaks have been identified at m/z5443, 7270, 7724, and 9888 for Bacillus subtilis, and at3603, 5496, 6800, 8858 and 9531 for Serratia marces-cens. The detection limits for the three target bacteriarange from 2.4×105 to 3.3×105 cfu·mL-1. We conclude

that the TE buffer approach can produce reliable data forrapid classification, high-resolution and highly sensitivedetection of bacteria.

Keywords Bacteria . MALDI-TOF MS . Protein ions .

Tris-EDTA buffer

Introduction

MALDI-TOF mass spectrometry is employed in microbi-ology to characterize specific peptides or proteins directlydesorbed from intact bacteria, viruses, and spores. Theability of monitoring ions over a broad m/z range, whichare unique and representative for individual bacteria andwhich correspond to biomarker constituents, forms thebasis of taxonomic identification of bacteria by MALDI-TOF MS with clinical relevance for the identification ofmany infectious diseases. It has long been recognized thatthe study of infectious diseases could not proceed withoutclear circumscription of the microbe. Hence from itsinception to the present time considerable effort has beendevoted to continually improving available methods formicrobial characterization. A number of different proce-dures have been developed in recent years that use massspectrometry for the direct determination of protein in acomplex mixture of biological origin. In particular, theapplication of MALDI mass spectrometry permits to obtainbiomarker profiles directly from unfractionated micro-organisms as bacteria, viruses, fungus cells and spores.This approach enables to detect, characterize and identifypeptides and proteins from intact bacteria and is applied inbiotechnology, cell biology and pharmaceutical research[1]. As biotechnology and microbiology examples, proteinexpression profiles from bacterial and eukaryotic cells and

Electronic supplementary material The online version of this article(doi:10.1007/s00604-011-0714-0) contains supplementary material,which is available to authorized users.

F. Ahmad :H.-F. Wu (*)Department of Chemistry, National Sun Yat-Sen University,Kaohsiung 80424, Taiwane-mail: hwu@faculty.nsysu.edu.tw

F. Ahmad :H.-F. WuCenter for Nanoscience and Nanotechnology,National Sun Yat-Sen University,Kaohsiung 80424, Taiwan

H.-F. WuDoctoral Degree Program in Marine Biotechnology,National Sun Yat—Sen University,Kaohsiung 80424, Taiwan

Microchim Acta (2012) 176:311–316DOI 10.1007/s00604-011-0714-0

cell-free extracts could be rapidly obtained by MALDI-TOF MS analysis [2]. The identification by MALDI-TOFMS of intact gram negative and positive bacteria, takendirectly from culture, provides a unique mass spectralfingerprint of the bacterial cell membrane [3]. However,reproducible sample preparation and mass spectra aredifficult to achieve in experiments that involve direct cellionization by MALDI and large variations can be found.Indeed, the sample preparation strategy adopted inMALDI-TOF MS of whole bacteria cells influences thedetection of signals with different masses. Saenz et al. [3]used MALDI-TOF MS to demonstrate the reproducibilityof bacterial spectra collected on different days in order todetect specific biomarkers. This work demonstrated thevalidity of the direct bacteria analysis by MALDI-TOFMS in obtaining consistent spectra from biologicalsamples over a period of time [4].

It is now apparent that after a significant incubationperiod in analytical research area, MALDI-TOF MS hasmatured into an analytical technique capable of confi-dently delivering information of clinical and industrialimportance [5]. Therefore, the aim of the present study isto develop the new technique as an improved method touse in the rapid screening and characterization ofpathogenic and non-pathogenic bacteria. We develop inmind that if successful, the method would support existingmethodologies, for detection of organism specific bio-marker molecules, which allow differentiation betweenbacteria to be made. This study not only establishes thecurrent protocols, data collection and analysis but alsoprovides data with improved mass resolution and sensitivityfor future analyses.

Experimental

Chemicals, materials, and bacteria

Chemicals purchased from Sigma (St. Louis, MO, USA,http://www.sigmaaldrich.com) included Ethylene diamine-tetraaceticacid (EDTA), Tris (hydroxyl methyl) aminomethane hydrochloride (Tris HCl), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA), tri-fluoro aceticacid (TFA; HPLC grade), acetonitrile (HPLC grade),methanol (HPLC grade). Deionized water (18.2 M Ω cm)obtained from a Milli-Q Plus pure water system (Millipore,Bedford, MA, USA, http://www.millipore.com) was auto-claved at 121 °C before use. The gram-positive andgram-negative bacterial strains used in this study werepurchased from the Bioresource Collection and ResearchCenter (BCRC, http://www.bcrc.firdi.org.tw), Hsinchu30062, Taiwan. Pure strains of bacteria were cultured onLuria Broth Agar (LBA) plates for 24 h at 37 °C. The

bacterial concentrations were determined by standard platecounting method [6]. All microbiological procedures werecarried out in a biosafety level 1 cabinet (NuAire,Plymouth, MN, USA, http://www.nuaire.com/).

Preparation of bacterial sample solutions

Bacterial strains were initially grown on Luria Brothagar (LBA) plates at 37 °C for 24 h. The bacterial cellswere harvested from LBA plates, and then suspended ineppendorf tube containing 2 mL of deionized water. Theeppendorf tube containing bacteria were vortexed for5 min. The bacterial cell suspensions were concentratedby centrifugation at 4,000 rpm for 5 min. Thesupernatant was poured off, and again 1.5 mL ofdeionized water (DI) was added to bacterial sediments.The bacterial solutions were then vortexed for 5 min toproper resuspend of intact bacteria. The washed bacterial cellswere collected by centrifugation (19,000 rpm) for 30 min, andthe supernatant was poured off and bacterial sediments weremixed with 1.5 mL Tris-Ethylendiaminetetraacetic acid(EDTA) buffer (10 mM Tris HCl, 1 mM EDTA, pH 7.5).The Tris-EDTA buffer was prepared by mixing 0.0078 g TrisHCl/5 mL DI H20 with 0.0014 g EDTA/5 mL DI H20. Tolyses the cells, bacterial cells and Tris-EDTA buffer reagentmixtures were incubated on vortex for 20 min at roomtemperature. Disrupted cells containing membrane fractionswere concentrated by centrifugation at 19,000 for 30 min atroom temperature. The collected biological samples weremixed with Tris-EDTA buffer and shaking for 10 min. Thisbiological sample was used for MALDI-TOF mass spec-trometry analysis, and no further cell lyses was undertaken.In the direct analysis, we used fresh harvested bacteria bymixing with DI water, and immediately loaded for MALDI-TOF mass analysis. The bacteria viz., Serratia marcescensBCRC10768, Escherichia coli BCRC12570 and Bacillussubtilis BCRC10029 were used separately to prepare thefinal cells suspension in Tris-EDTA buffer according toabove described method. The each bacterial species wasadjusted separately to concentrations of 105, 106 and 107 cfu.mL-1 using Milli-Q grade water. Fresh harvested bacteriawere used in this experiment. Scheme 1 represents themethodology applied in this study.

Bacterial samples for MALDI-TOF MS

Before MALDI-TOF MS analysis, the MALDI target plateswere washed with Milli-Q grade water, treated withmethanol, and allowed to dry at room temperature. Thebacterial cells fixed in Tris-EDTA buffer was suspended byvortexing and 0.5 μL of each was immediately transferredto wells of a 96-well MALDI target plate. Each well wasimmediately overlaid with 0.5 μl matrix solutions (50 mM,

312 F. Ahmad, H.-F. Wu

Sinapinic acid). The sinapinic acid was freshly prepared asa saturated solution in 3:1 acetonitrile : water containing0.1% trifluoroacetic acid (TFA). The bacterial samplesmixtures were allowed to dry at room temperature inambient air prior to MALDI-TOF MS analysis.

Instrumentation

All mass spectra were generated in the positive ion modeusing a MALDI-TOF MS (Microflex Bruker Daltonics,Bremen, Germany); 337 nm of a nitrogen laser was usedfor irradiation of the analytes. The accelerating voltageswere set at 20 kV. All mass spectra (150 laser shots for perspectrum) were generated in the linear mode for proteins.Other parameters were set the same as those describedpreviously [7]. All experiments were carried out at leastthree times in order to check the reproducibility of thecurrent approach.

Results and discussion

Rapid extraction of protein biomarkers from bacterialsamples and other microorganisms may be achieved bythe use of strong acid or organic solvents [8, 9]. Anautomated system would be expected to provide adequate

well organized method and conditions for effective celllyses and also for biomarker ionization. Different protocolshave been developed for various sample types, rangingfrom methods applicable to whole cell lyses of bacteria[10]. The MALDI mass spectrum of the bacterial sampleused in this work was obtained using Tris-EDTA bufferapproach (TEBA) as shown in Fig. 1, 2 and 3. The methodvariables than reported methodology of earlier researchers,that were evaluated appeared to improve the quality (i.e.,resolution, quantity of peaks, intensity, and mass range ofproteins observed) of the bacteria spectra. This result wasverified using the final set of experimental conditionsdescribed in this work as the “optimum” method forMALDI -TOF MS analysis of whole cells prepared withTris-EDTA buffer. In this study, we reported first timemany marker peaks with the application of TEBA. With thebest of our knowledge, the current applied method is notreported by others researchers. This method is very simple,rapid and easy to perform in biological laboratories. Thisobservation indicates that cell growth handling prior toanalysis is an important factor that needs systematicexamination to adequately scope the potential utility ofMALDI-TOF MS for rapid and sensitive analysis.

In the present study, Fig. 1, 2 & 3 depict mass spectralinformation were obtained from whole bacterial cellssuspension for S. marcescens, E. coli and B. subtilis isolate.

Scheme 1 Schematic represen-tation for the Tris-EDTAbuffer approach used in theexperiments to determinethe bacteria via whole cellsMALDI-TOF MS inbiological sample

High-resolution MALDI-TOF mass spectrometry of bacterial proteins 313

The number of reproducible biomarker protein peaks of S.marscens were observed (Fig. 1a,b,c). We selected threedifferent cells concentration to check the sensitivity ofpresent approach. The results revealed that the appliedapproach was successfully produced the mass peaks, evenat the low bacterial concentrations ~105 cfu.mL-1. Thefollowing biomarker peaks at m/z 3603, 5496, 6800, 8858and 9531 were identified from S. marcescens using currentTEBA. The identified mass peaks are shown in Fig. 1. Therepresentative mass spectrum of intact E. coli cells is shownin Fig. 2, where a substantial number of peaks were observedin the mass range 3,000 to 15,000 Da. The E. coli spectrumobtained in this experiment is qualitatively similar to thosereported by earlier researchers [11–13]. Most prominently,many of the intense peaks observed earlier are also found inthis study. The results indicated that mass peaks depend onthe preparation of biological samples, because the directanalysis was showed the less number of peaks with lowintensity (Fig. S2) as compared to mass peaks obtainedwith application of TEBA. The prepared samples of E.coli (2.44×105 cfu.mL-1) showed the signals at m/z 3636,5466, 8296 and 9192. While higher concentration ofprepared samples of E. coli were depicted more strong

mass peaks with many biomarkers. The peaks at 3636,4871, 5466, 5750, 6547, 7274, 8296, 9192 & 9742; and3636, 3933, 4871, 5466, 5750, 6315, 6547, 7274, 7654,8296, 8706, 9192, 9742, 10938 &13730 Da wereobserved at 2.44×106 and 2.44×107 cfu.mL-1 respective-ly. The 9742 and 4,871 Da features appear to be the singlyand doubly charged ions of the same protein, which areshown in panel b & c of Fig. 2. These results confirm thatour approach is a successful one for generating strong masspeaks that allow an examination of the intact bacteria.

To check strong mass peaks with good intensities as wellas to isolate and we further characterize the proteins fromthe B. subtilis, we used the TEBA to prepare the bacterialsamples combined with MALDI analysis to reveal thepeaks of bacterial protein content. Panels a-c of Fig. 3represent the mass spectra obtained after preparing thebacteria samples to selectively concentrate B. subtilis atconcentrations of 2.86×105, 2.86×106, and 2.86×107 cfu.mL-1, respectively. The ions at m/z 5443, 6427, 7724, 7953,9470, and 9888 which represent B. subtilis, appearedreproducibly in Fig. 3b and c. When the concentration ofB. subtilis was decreased to 2.86×105 cfu.mL-1, only the

Abu

ndan

ce

a

b

c

Fig. 2 Mass spectra obtained from MALDI-MS analysis of proteinextracts from E. coli BCRC12570 at different cells concentrations. a2.44×105 , b 2.44×106 , and c 2.44×107 cfu.mL-1. The bacterial cellssuspensions were prepared in Tris-EDTA buffer

Abu

ndan

cea

b

c

Fig. 1 Mass spectra obtained from MALDI-MS analysis of proteinextracts from S. marcescens BCRC10768 at different cells concen-trations. a 3.30×105 , b 3.30×106 , and c 3.30×107 cfu.mL-1. Thebacterial cells suspensions were prepared in Tris-EDTA buffer

314 F. Ahmad, H.-F. Wu

ions at m/z 5443, 6427 and 7724 appear in the massspectrum (Fig. 3a). These results indicate that the currentapproach is capable of producing the strong mass peaks oftarget bacteria from solutions. In comparison, the directanalysis was not able to produce strong peaks, which areshown in Fig. S3 of supporting information. The aboveapplied method is very simple and rapid since the MALDItarget plate holds 96 sample wells, which may be analyzedin short period. In this study, we compared this approach todirect analysis. The treated bacterial cells with Tris-EDTAbuffer exhibit strong mass peaks with more signal enhance-ments, which is due to the disruption of membrane proteinsin solution. The results obtained from both the Tris-EDTAbuffer treated bacterial cells and the aqueous bacterial cellsmethods were compared and the results are presented inFig. 1,2 & 3 and Fig. S1, S2 & S3. The main differencebetween the both methods is that bacterial cells treated withTris-EDTA buffer have demonstrated to release moremembrane proteins in solution and show more number ofmass peaks with strong signal intensity. This can be provedby two facts. First, the signal intensity for Tris-EDTAbuffer treated bacteria is always higher than that of direct

analysis of bacteria in all spectra when equal concentrationsof selected bacteria were analyzed in the sample solutions.Secondly, the limit of detection of bacterial treated cellswith Tris-EDTA buffer is higher than that of aqueousbacterial cells suspension used as direct analysis.

One of the ultimate goals of the MALDI-TOF MSmethod in bacterial identification is to quickly differentiateand even accurately identify closely related species.Existing research results have convincingly demonstratedthe capability of differentiating closely related strains of thebacteria. Mass spectral data acquired under specific conditionswill be generated and stored, such that bacterial cells in fieldand / or unknown samples can be automatically detectedby comparison of newly recorded spectra and observedbiomarkers with library data. Data provided from samplepreparation, can also be entered into the database. Researchand development efforts are presently underway to apply MS/MS technique for selecting the biomarkers. Effort to optimizethe experimental conditions pertaining both to bacterialgrowth and protein extraction presently being pursuedtogether with improvement of the measured mass accuracyduring the MALDI-TOFMS analysis. However, there are stillmany other unidentified ions appearing in the mass spectra,the potential biomarker ions representing specific bacterialstrains can be readily identified in the mass spectrabased on the potential biomarkers ions listed in Table 1.

Abu

ndan

cea

b

c

Fig. 3 Mass spectra obtained from MALDI-MS analysis of proteinextracts from B. subtilis BCRC10029 at different cells concentrations.a 2.86×105 , b 2.86×106 , and c 2.86×107 cfu.mL-1. The bacterialcells suspensions were prepared in Tris-EDTA buffer

Table 1 The biomarker protein peaks (m/z value) of selected targetbacteria were detected using Tris-EDTA buffer approach coupled withMALDI-TOF MS

Bacterial strains Detection of biomarker proteinions (m/z value) in different cellsconcentrations (cfu.mL-1)

~105 cfu.mL-1 ~106 cfu.mL-1 ~107 cfu.mL-1

Serratia marcescens(BCRC 10768)

9,531 3,603 3,603

– 6,800 5,496

– 9,531 6,800

– – 8,858

– – 9,531

Escherichia coli(BCRC12570)

3,636 3,636 3,636

5,466 5,466 5,466

9,192 5,750 5,750

– – 6,315

– 6,547 6,547

– 7,274 7,274

– 9,192 9,192

– 9,742 9,742

Bacillus subtilis(BCRC10029)

5,443 5,443 5,443

7,724 7,724 7,270

– 9,888 7,724

– – 9,888

High-resolution MALDI-TOF mass spectrometry of bacterial proteins 315

The results look quite promising; this approach seemsvery suitable to be used in characterization of thesesbacterial strains in clinical, environmental and biologicalsamples. Satisfactory, reproducible bacterial mass spectracan be demonstrated with the examples of S. marcescens,E. coli and B. subtilis.

Conclusions

We have identified possible biomarkers for three bacterialstrains in this study involving adopted cell lyses procedure asdescribed in experimental section. The bacterial strains arereadily differentiated based on these potential biomarkers. Tobest of our knowledge, this paper provides first example ofusing Tris-EDTA buffer solution combined with MALDI-TOF mass spectrometry based proteomic strategy to success-fully differentiate the bacteria. It is important to note thatapplication of Tris-EDTA buffer was produced the reproduc-ibility of spectra with number of strong mass peaks. The massspectra obtained gave profiles representative of the molecularweight (3,000–15,000 Da) components of cells extract. Fromthe mass spectra it was demonstrated that differentiation ofindividual bacteria down to the level of specific strains couldbe accomplished. We believe that the Tris-EDTA bufferapproach can be utilized to demonstrate the rapidly detectablebiomarkers ions for identification of bacteria with highresolution and high sensitivity.

Acknowledgement This work was supported by the NationalScience Council, Taiwan under the grant NSC: 98-2113-M-110-007-MY3.

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