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Combination of Capillary Isoelectric Focusing in a Tapered Capillary with MALDI-TOF MS for Rapid and Reliable Identification of Dickeya Species from Plant Samples Marie Horká,*,† Jiří Šalplachta,† Pavel Karásek,† Anna Kubesová,† Jaroslav Horký,‡ Hana Matoušková,‡ Karel Šlais,† and Michal Roth† †

Institute of Analytical Chemistry of the ASCR, v. v. i., Veveří 97, 602 00 Brno, Czech Republic Division of Diagnostics, State Phytosanitary Administration, Šlechtitelů 23, 779 00 Olomouc, Czech Republic

‡

ABSTRACT: This study was undertaken to investigate feasibility of a combination of capillary isoelectric focusing (CIEF) in a tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) for a rapid and reliable identification of bacteria taken from plant-tissue-containing samples. Eight strains representing different species of the genus Dickeya were selected on the basis of close proximity of their isoelectric points: D. chrysanthemi, D. chrysanthemi bv. parthenii, D. chrysanthemi bv. chrysanthemi, D. dadantii, D. paradisiaca, D. solani, D. dif fenbachiae, and D. dianthicola. Because the Dickeya species (spp.) cannot be easily discriminated from each other when CIEF is performed in a cylindrical FS capillary (commonly used in CIEF) even if a narrow pH gradient is used, a tapered FS capillary was employed instead, which enabled satisfactory discrimination of the examined bacteria due to enhanced separation efficiency of CIEF in the tapered FS capillary. CIEF in the tapered FS capillary was also successfully used for the detection and characterization of Dickeya spp. in a plant-tissue-containing sample. Then an off-line combination of CIEF with MALDI-TOF MS was employed for rapid and reliable identification of Dickeya spp. in the planttissue-containing sample. It was found that the presence of plant tissue did not affect the results, making the proposed procedure very promising with respect to the fast and reliable detection and identification of bacteria in plant-tissue-containing samples.

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In this study, the advantages of tapered fused silica (FS) capillary over the cylindrical FS capillary are demonstrated on the CIEF separation of bacteria within the genus Dickeya. Dickeya species (spp.), which are pectinolytic Gram-negative bacteria, are broad-host-range pathogens that cause diseases on numerous plants including many economically important crops.8,22−25 These bacteria produce a variety of cell-walldegrading enzymes that allow infiltration and maceration of plant tissues on which the bacteria feed.26 Because the Dickeya spp. are responsible for significant losses in agricultural production, reliable and accurate identification methods are of great interest and importance. Current approaches to the detection and identification of Dickeya spp. include cultural, serological, and molecular methods. However, the use of serology, including ELISA27and and immunofluorescence, is problematic with respect to the specificity and sensitivity. Nowadays, the Dickeya spp. are usually detected by polymerase chain reaction (PCR) methods.23,27Although these methods can detect a pathogen, they often

apillary isoelectric focusing (CIEF) represents an effective analytical tool for differentiation and characterization of many microorganisms according to their isoelectric points, pI’s.1−9 Unlike other electrophoretic techniques,5,6,10−12 pI values of microorganisms are independent of the properties of the external environment. A CIEF discrimination of microbes with close pI values is rather difficult, especially when the separation is performed in a wide pH gradient.13−15 This can partly be solved by separation in a very narrow pH gradient.16 However, CIEF in such a pH gradient is not easily applicable in common laboratory practice. A possible way to overcome this problem is the use of a tapered capillary with continuous decrease of the inner diameter (i.d.) toward the detection point.17−19 In CIEF carried out in a tapered capillary with the inlet cross-section three times larger than the cross-section at the detection window, several times higher resolutions of corresponding peak pairs were obtained20 as compared to those achieved with a conventional cylindrical capillary. The tapered capillary was prepared with supercritical water (SCW) which is able to modify the inner surface of silica-based material.20,21 Moreover, SCW does not contaminate the treated surface with any residual impurities which is an essential requirement for highly selective and highly efficient biomolecular separations. © 2013 American Chemical Society

Received: March 28, 2013 Accepted: June 14, 2013 Published: June 14, 2013 6806

dx.doi.org/10.1021/ac4009176 | Anal. Chem. 2013, 85, 6806−6812

Analytical Chemistry

Article

controlled by serial dilution and plating of 100 μL of the suspension on nutrient agar. After the cultivation at 28 °C for 24 h, the colonies were counted. Safety. According to international standards, the strains of phytopathogenic bacteria used may not cause human and/or animal diseases, might be a hazard in a laboratory working with plant material, but are unlikely to spread to the community. Laboratory exposure produces a risk of contamination, and effective prophylaxis or effective treatment is available. Fabrication of a Tapered FS Capillary. Tapered FS capillaries were prepared from commercially available cylindrical FS capillaries (100 μm i.d., 360 μm o.d., Agilent Technologies, Waldbronn, Germany, part no. 160-2634-10) by etching with supercritical water in our original apparatus,21 employing a procedure similar to that described previously.20 CIEF Equipment and Procedures. CIEF experiments were carried out using a laboratory-made apparatus14 at a constant voltage of −20 kV on the side of the detector supplied by a Spellman (Plainview, NY) CZE 1000 R high voltage unit. Separations were performed using both cylindrical and tapered FS capillaries. The total length of the cylindrical capillary, 100 μm i.d. and 360 μm o.d., was 300 mm (Agilent Technologies); the effective length to the detection window was 200 mm. The total length of the tapered capillary, inlet i.d. 175 μm, outlet i.d. 100 μm, 360 μm o.d., was 480 mm; the effective length to the detection window was 300 mm. The following conditions were identical for both types of separation capillaries. The ends of the capillary and the electrodes were placed in 3mL glass vials filled with an anolyte or a catholyte. On-column UV−vis detector LCD 2082 (Ecom, Prague, Czech Republic), connected to the detection cell by optical fibers (Polymicro Technologies, Phoenix, AZ), was operated at 280 nm. Light absorption (optical density) of the bacterial samples was measured by use of a Beckmann Instruments (Palo Alto, CA) DU 520 UV−vis spectrophotometer operating at 550 nm. Sample injection was performed by siphoning action as described in our previous paper.13 Cell clusters were deagglomerated by sonication in a Sonorex (Bandelin Electronic, Berlin, Germany) ultrasonic bath and then vortexed using a Yellowline TTS 3 Digital Orbital Shaker (IKA Works, Wilmington, DE) immediately before injection of the bacterial sample into the capillary. The sonication was performed at 25 °C and 35 kHz for 1 min for each sample. Detector signals were acquired and processed with a Clarity Chromatography Station (ver. 2.6.3.313, DataApex, Prague, Czech Republic). For CIEF separations, 4 × 10−2 mol L−1 sodium hydroxide and 0.1 mol L−1 orthophosphoric acid, both with addition of 3% (v/v) EtOH and 0.5% (w/v) PEG 10000, were used as catholyte and anolyte solutions, respectively. Before each CIEF run, the capillaries were rinsed with EtOH for 5 min and then back-flushed with catholyte for 5 min. For this purpose, a single-syringe infusion pump (Cole-Parmer, Vernon Hills, IL) equipped with a 100-μL syringe (SGE Analytical Science, Victoria, Australia) was used at a flow rate ranging from 3 to 20 μL min−1. Segmental injection into the capillary was employed in CIEF analysis.13,14 Solutions necessary for the CIEF run (including the sample) were injected in three consecutive segments. The first was the segment of spacers, a solution of selected simple ampholytic electrolytes, 15 × 10−5 mol L−1, dissolved in 4 × 10−2 mol L−1 NaOH. The second segment was the cell suspension of the examined Dickeya spp., suspension of potato tubers, or suspension of potato tubers spiked with the examined bacteria. For CIEF experiments, all these samples

fail in the case of bacterial classification. Recently, matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a new tool for rapid and effective characterization of bacteria.28−31 Identification of bacteria is based on the unique mass/charge (m/z) fingerprints of cell components, especially peptides and proteins, which are characteristic of individual bacterial spp. or subspecies (subsp.). The MALDI-TOF MS approach was also used for the classification or detection of bacteria within genus Erwinia,32,33which belongs (as well as Dickeya spp.) to the family Enterobacteriaceae.23 The aim of this study was to demonstrate the potential and capabilities of CIEF in the tapered capillary for effective and unambiguous UV detection and identification of closely related bacteria. Feasibility of combination of CIEF in tapered capillary with MALDI-TOF MS for reliable identification of bacteria in plant-tissue-containing samples was also investigated.

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EXPERIMENTAL SECTION Chemicals. Poly(ethylene glycol), Mr = 10000 (PEG 10000) was obtained from Aldrich (Milwaukee, WI). The high resolution ampholyte, pH 2−4, ampholyte pH 3−4.5, 2morpholinoethanesulfonic acid monohydrate, 3-morpholinopropanesulfonic acid, and N-[tris(hydroxymethyl)methyl]-3amino-2-hydroxypropanesulfonic acid were obtained from Fluka Chemie (Buchs, Switzerland). The solution of synthetic carrier ampholytes (Biolyte, pH 3−10) was obtained from BioRad Laboratories (Hercules, CA). N-(2-Acetamido)-2-aminoethanesulfonic acid and 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid were obtained from Merck (Darmstadt, Germany). L-Aspartic acid was obtained from LOBA Chemie (Wien, Austria). Other spacers (tartaric, malic, formic (FA), succinic, acetic, pivalic, glutamic, and nicotinic acids), acetonitrile (ACN), trifluoroacetic acid (TFA), and ethanol (EtOH), were purchased from Sigma (St. Louis, MO). The specifications of the spacers used,34,35 all simple ampholytic electrolytes, are described elsewhere.14 The low-molecular-mass pI markers, pI = 2.0, 2.7, 3.7,36 4.0,37 and 4-morpholinylacetic acid36 were synthesized at the Institute of Analytical Chemistry of the ASCR. 3,5-Dimethoxy-4-hydroxycinnamic acid (SA) and calibration mixture ProMix2 were purchased from LaserBio Laboratories (Sophia-Antipolis Cedex, France). All chemicals were of analytical or MS grade. Bacterial Strains and Growth Conditions. The strains of Dickeya spp. and subsp. included in this study, D. chrysanthemi CFBP 3330, D. chrysanthemi bv. parthenii CFBP 2117, D. chrysanthemi bv. chrysanthemi PRI 2119, D. dadantii PRI 3332, D. paradisiaca PRI 2128, D. solani PRI 2019, D. dif fenbachiae PRI 1259, and D. dianthicola PRI 3331, were obtained from La Collection Française de Bactéries associées aux Plantes (CFBP), Angers, France, or from Plant Research International (PRI), Wageningen, The Netherlands. The bacterial strains from culture collections were cultivated on nutrient agar at 28 °C for 24 h. Potato tubers (1 g) were homogenized in aseptic conditions with mortar and pestle in sterile demineralized water (5 mL), streaked on modified crystal violet pectate selective medium (CVP),38 and incubated at 20 °C and 27 °C for 48−72 h to check sterility of the sample. A DEN-1 McFarland densitometer (Biosan, Riga, Latvia) was used for measurement of the cell concentration in the suspensions. The visual comparison of organism suspensions to turbidity standards is an accepted method of estimating organism densities. The numbers of microorganisms in the reference samples were also 6807



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RESULTS AND DISCUSSION CIEF of Dickeya spp. in Cylindrical and Tapered FS Capillaries. In our preliminary experiments, different Dickeya

were resuspended in demineralized water with or without addition of 1% (w/v) PEG 10000. The cell concentration was adjusted to 1 × 108 cells mL−1 and to 1 × 107 cells mL−1 for cylindrical FS capillary and tapered FS capillary, respectively. The third segment was a 5% (w/v) aqueous solution of the commercial carrier ampholytes Biolyte pH 3−10, ampholytes pH 2.0−4.0, and pH 3.0−4.5, mixed in the ratio 1:5:2. This segment also contained low-molecular-mass pI markers (pI 2.0, 2.7, and 4.0; concentration of individual pI markers 25 μg mL−1) for monitoring the pH gradient used (2.0−4.0). Injection time of the spacers segment, the sample segment, and the segment of carrier ampholytes and pI markers was 25 s, 10 s, and 35 s, respectively. Bacteria separated in the tapered capillary were pushed out of the cathodic end of the capillary using a single-syringe infusion pump attached to the anodic end of the capillary, at a flow rate of 50 nL s−1. As soon as the zone of analyzed bacterium was detected, the content of capillary between the detector and the cathodic end was pushed out of the capillary in 28 s (the zone of bacterium reached the end of the capillary in 28 s after the detection). Thereafter, a small drop (about 0.35 μL in 7 s) containing the analyzed bacterium was deposited manually on a MALDI sample plate (the droplet simply touched the sample spot of the plate) and left to dry at room temperature. MALDI-TOF Mass Spectrometry. For MALDI-TOF MS experiments, collected cells of each bacterial strain were resuspended in deionized water, and their concentration was adjusted to 1 × 108 cells mL−1. Fifty microliters of the bacterial suspension was centrifuged at 3000g for 3 min, supernatant was removed, and 50 μL of 70% (v/v) EtOH was then added to the pellet. After brief shaking, the suspension was centrifuged again at 3000g for 3 min, the supernatant was removed, and the pellet was resuspended in 50 μL of ACN/FA solution (ACN/5% FA, 1:1, v/v). This bacterial suspension was mixed with SA solution (16 mg mL−1 in ACN/0.1% TFA, 3:2, v/v) in 1:1 ratio immediately before MS analysis. The resulting mixture (0.8 μL, which represents approximately 4 × 104 cells) was spotted on the sample plate previously overlaid with the SA solution and left to dry at room temperature. A bacterial sample collected from the CIEF run and spotted on the sample plate was overlaid with the SA solution. Finally, each sample spot was overlaid with ACN/H2O solution (ACN/H2O, 3:2, v/v) to get a more homogeneous surface, which enhanced the reproducibility of the measured mass spectra. MS experiments were performed on a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA) operating in a linear mode. Accelerating voltage was set at 20 kV. Each mass spectrum was automatically acquired by accumulation of 1400 laser shots (70 laser shots delivered at a single point, 20 different points randomly selected within given sample spot) in linear positive ion mode. To evaluate the reproducibility of the measured data, the bacterial samples (both cellular suspension and the sample collected from CIEF run) were spotted in quadruplicates on the sample plate. Each sample spot was analyzed twice. Calibration of mass spectra was performed externally with a protein calibration mixture containing equine cytochrome c (12 360 Da), equine myoglobin (16 951 Da), and bovine trypsinogen (23 981 Da). Both singly and multiply charged ions were used for calibration to cover the required m/z range. Recorded mass spectra were processed using Data Explorer (ver. 4.6, Applied Biosystems). Mass accuracy for protein molecular weight determination was found to be better than 0.16% for all the bacterial samples.

Figure 1. CIEF of Dickeya spp. with mutually close pI values in the pH gradient, pH range 2.0−4.0, in the cylindrical FS capillary. The examined bacteria were resuspended in demineralized water with addition of 1% (w/v) PEG 10000 to a concentration of 1 × 108 cells mL−1.

Table 1. Migration Time t and pI of Different Dickeya spp.a FS capillary cylindrical species D. chrysanthemi bv. parthenii D. chrysanthemi D. chrysanthemi bv. chrysanthemi D. dadantii D. solani D. paradisiaca D. dianthicola D. dif fenbachiae approximate RSD

tapered

t [min]

RSD [%]

pI

t [min]

RSD [%]

pI

11.75

1.4

2.70

16.20

1.7

2.69

11.51 10.57

1.5 1.6

2.80 3.16

15.71 14.23

1.7 1.8

2.81 3.15

10.43 10.38 10.25 10.12 10.00

1.4 1.6 1.4 1.5 1.4 1.5

3.20 3.22 3.30 3.35 3.40

13.91 13.80 13.49 13.25 13.02

1.7 1.9 1.7 1.8 1.8 1.8

3.19 3.24 3.31 3.34 3.41

a

The data were obtained from the separate CIEF of individual species which are depicted in Figures 1 and 2. Each measurement was repeated at least 10 times.

spp. were analyzed by CIEF in cylindrical FS capillaries over a wide pH gradient, 3.0−10.0, to find bacterial species with close pI values. On the basis of these experiments, eight bacterial strains belonging to various spp. and subsp. of genus Dickeya were selected and analyzed by CIEF in both cylindrical and tapered FS capillaries. At first, each bacterial strain was analyzed in a cylindrical capillary over the pH gradient 2.0−4.0. The length of the cylindrical capillary and the consequent time for the separation of microorganisms is limited with respect to the 6808



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Figure 2. CIEF of Dickeya spp. with mutually close pI values in the pH gradient, pH range 2.0−4.0, in the tapered FS capillary. The examined bacteria were resuspended in demineralized water to a concentration of 1 × 107 cells mL−1.

minimal thermal shock on the cells due to the Joule heating effect. The concentrations of electrolyte additives used, 3% (v/ v) EtOH and 0.5% (v/v) PEG 10000, were appropriate for preventing the bacteria from adsorbing on the inner surface of capillary. These additives also decreased the electroosmotic flow. CIEF separation of different Dickeya spp. in cylindrical FS capillaries is depicted in Figure 1. To avoid a coincidence of the migration times of bacteria and pI markers, the pH gradient was traced with only three pI markers, pI 2.0, 3.7, and 4.0, and the eight bacterial strains were analyzed in two separate runs (A and B, four strains each). The pI values of the examined bacteria were calculated from the migration times and the pI values of the pI markers. The pI values determined via CIEF in cylindrical FS capillary are listed in Table 1. Each bacterial strain was analyzed 10 times, and the relative standard deviation (RSD) of the migration times was about 1.5%. As our experimental results show (Figures 1A,B), the pI values of some bacteria are very close to each other: D. chrysanthemi bv. chrysanthemi and D. dadantii (Δ pI = 0.04), D. chrysanthemi and D. chrysanthemi bv. parthenii (Δ pI = 0.1), D. dif fenbachiae and D. dianthicola (Δ pI = 0.05), D. paradisiaca and D. solani (Δ pI = 0.08). Because of the subtle differences in the pI values, these bacteria cannot be easily discriminated from each other when the CIEF is performed in a cylindrical FS capillary. Moreover, the resolution decreased with the decreasing number of cells injected into the cylindrical capillary. Although specific experimental conditions could provide a better discrimination of bacteria with close pI values,1,16,39 the successful procedure is very difficult to achieve and is not easily applicable in common laboratory practice. Utilization of the tapered FS capillary21 in CIEF of bacteria seems to be very advantageous. All of the above-mentioned CIEF experiments were also performed in a tapered capillary to discriminate bacteria with close pI values. The experimental

Figure 3. CIEF in the pH gradient, pH range 2.0−4.0: (A) analysis of the suspension of potato tubers in the cylindrical FS capillary, (B) analysis of the suspension of potato tubers in the tapered FS capillary, (C) analysis of the suspension of potato tubers spiked with D. solani and D. paradisiaca in the tapered FS capillary. Concentration of each bacterium was 1 × 107 cells mL−1. (D) Analysis of the suspension of potato tubers spiked with D. solani (1 × 108 cells mL−1) in the tapered FS capillary with indication of individual separated zones of the injected sample: part 1, the zone is already outside the capillary at the time of detection; part 2, waste (28 s); part 3, zone of the bacterium collected and deposited on the sample plate (7 s); part 4, waste. The dashed line marks the beginning of the hydrodynamic extrusion, performed at a flow rate of 3 μL min−1, of the sample zone from the cathodic end of the capillary.

conditions, including the pH gradient used, were the same as for CIEF in a cylindrical FS capillary except for the cell concentration. For CIEF experiments performed in a tapered FS capillary, the cell concentration was adjusted to 1 × 107 cells mL−1. An example of CIEF separation of the examined Dickeya spp. in a tapered FS capillary is shown in Figure 2. The use of a tapered FS capillary enabled a satisfactory discrimination of the examined bacteria (Figure 1 vs Figure 2). Similarly to CIEF in the cylindrical FS capillary, the pH gradient was traced with only three pI markers (pI 2.7, 3.7, and 4.0) to avoid coincidence of the migration times of separated bacteria and pI markers. Very good reproducibility was achieved by CIEF in the tapered FS capillary; see Table 1. The RSDs of the migration times, calculated from 10 independent analyses of each bacterial strain, were about 1.8%. The maximum observed difference in pI values of 10 independent analyses of individual bacteria was 0.01 units of pI. Within the limits of measurement error, the pI 6809



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Figure 4. MALDI-TOF MS spectra acquired in linear positive ion mode. (A) Cellular suspension of D. solani, 1 × 108 mL−1. (B) D. solani collected from the CIEF of potato tubers spiked with the examined bacteria (separation in the tapered FS capillary, see Figure 3D).

values of the examined bacteria obtained from CIEF in tapered or cylindrical FS capillaries were comparable. CIEF of Potato Tubers Spiked with Bacteria. To assess the capability of CIEF to detect and characterize bacteria taken from plant material, a suspension of potato tubers spiked with the examined bacteria was analyzed in this study. Both cylindrical and tapered FS capillaries were used for this purpose, and the results are shown in Figure 3A and 3B. The detected signals of the suspension of potato tubers (background noise) were approximately 10 times higher in the cylindrical FS capillary compared to that in the tapered FS capillary. These results demonstrate that the detection of bacteria taken directly from plant material is quite difficult in the case of CIEF in the cylindrical FS capillary. On the contrary, the use of the tapered FS capillary enabled detection and characterization of the bacteria. An example of CIEF in the tapered FS capillary of potato tubers spiked with D. solani and D. paradisiaca (concentration of individual bacteria 1 × 107 cell mL−1) is depicted in Figure 3C. The presence of potato tubers did not affect the migration times of the analyzed bacteria. Each analysis was performed 10 times, and the RSDs of the migration times were less than 1.7%. To provide a better illustration, the CIEF results in Figure 3 are shown without the pI markers. Combination of CIEF in the Tapered Capillary with MALDI-TOF MS. Feasibility of the combination of CIEF

separation in the tapered FS capillary with MALDI-TOF MS for unambiguous identification of bacteria in plant-tissuecontaining samples was also investigated in this study. At first, the cell suspension of each examined bacteria was analyzed by MALDI-TOF MS to get mass spectra of the individual bacterial strains. An example of a MALDI-TOF mass spectrum of the examined D. solani is shown in Figure 4A. The figure shows characteristic mass fingerprints of the analyzed bacterium (approximately 4 × 104 cells were deposited on each sample spot of the sample plate) within the 2500 to 20000 m/z range. The subsequent experiments were performed using an off-line combination of CIEF in the tapered FS capillary with MALDITOF MS. A suspension of potato tubers spiked with the examined bacteria (1 × 108 cells mL−1) was subjected to CIEF (ca. 40 nL of the bacterial sample was injected into the capillary). The bacterium zone was then collected (see Figure 3D), deposited on the sample plate (approximately 4 × 103 cells were deposited on the sample spot in this case), and analyzed by MALDI-TOF MS as described in the Experimental Section. An example of MALDI-TOF mass spectrum of D. solani collected from the CIEF run is shown in Figure 4B. It was found that most of the mass signals detected in the mass spectra of cellular suspension of the individual bacteria were also detected in the corresponding mass spectra of the bacteria collected after the CIEF separation as can be seen in Figure 4. Regarding the examined D. solani, one major peak at m/z 6810



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14418 was found in the acquired mass spectra of the bacterial sample analyzed before and after CIEF. This peak was also found as the dimer and doubly charged and triply charged ions in the mass spectra. Furthermore, mass signals at m/z 5870, 6225, 6820, 7030, 8875, 9070, 9275, 9530, 10410, 10650, 10930, 11710, 12290, and 17320 were detected in the mass spectra of both samples of D. solani (Figure 4). However, some peaks are missing and some additional peaks (within the whole mass range recorded) are observed for the sample collected from the CIEF analysis. Different cell concentrations can play a role in this respect. This explains why some mass signals were not detected in the mass spectra of the sample collected from the CIEF run. Moreover, the mass signals found only in the mass spectra of the sample collected from CIEF most probably come from the potato tubers. Nevertheless, our data have proven that the combination of CIEF with MALDI-TOF MS can be used for fast (about 30 min for the entire analysis) and reliable identification of bacteria within the genus Dickeya in plant-tissue-containing samples.

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CONCLUSION In this study, feasibility of the combination of CIEF in a tapered FS capillary with MALDI-TOF MS for reliable identification of Dickeya spp. in plant-tissue-containing samples was investigated. At first, the examined bacteria were analyzed by CIEF in both cylindrical and tapered FS capillaries. Owing to better peak resolution, the use of the tapered FS capillary enabled satisfactory discrimination of Dickeya spp. in contrast to the cylindrical FS capillary (the analyses were performed over the same pH gradient). Moreover, the resultant pI values of the examined bacteria determined by CIEF in the tapered FS capillary were comparable with those obtained by CIEF in the cylindrical FS capillary. In addition, it was also found that CIEF in the tapered FS capillary can also be used to detect and characterize bacteria within the genus Dickeya taken from plant material, which is another important benefit of the tapered FS capillary. The presence of plant tissue affected neither migration times nor pI values of the analyzed bacteria. Finally, combination of CIEF with MALDI-TOF MS was successfully used for reliable identification of Dickeya spp. taken from plant material. This procedure is effective and very fast because the entire analysis was completed within 30 min. The results presented in this study are very promising, and the proposed procedure based on the off-line combination of CIEF in the tapered FS capillary with MALDI-TOF MS could solve the problem of reliable detection and identification of bacteria in plant-tissue-containing samples.

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AUTHOR INFORMATION

Corresponding Author

*Tel. +420 532 290 221. Fax: +420 541 212 113. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of the Interior of the Czech Republic (Grant VG20112015021), by the Czech Science Foundation (Grant P106/12/0522), and by the Academy of Sciences of the Czech Republic (Institutional Support RVO:68081715). H.M. took the photos in the TOC/ abstract graphic. 6811



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(38) Pérombelon, M. C. M.; Burnett, E. M. Potato Res. 1991, 34, 79− 85. (39) Righetti, P. G. J. Chromatogr., A 2004, 1037, 491−499.

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