A rapid biosensor for viable <Emphasis Type="Italic">B. anthracis</Emphasis> spores

A simple membrane-strip-based biosensor assay has been combined with a nucleic acid sequence-based amplification (NASBA) reaction for rapid (4 h) detection of a small number (ten) of viable B. anthracis spores. The biosensor is based on identification of a unique mRNA sequence from one of the anthrax toxin genes, the protective antigen (pag), encoded on the toxin plasmid, pXO1, and thus provides high specificity toward B. anthracis. Previously, the anthrax toxins activator (atxA) mRNA had been used in our laboratory for the development of a biosensor for the detection of a single B. anthracis spore within 12 h. Changing the target sequence to the pag mRNA provided the ability to shorten the overall assay time significantly. The vaccine strain of B. anthracis (Sterne strain) was used in all experiments. A 500-L sample containing as few as ten spores was mixed with 500 L growth medium and incubated for 30 min for spore germination and mRNA production. Thus, only spores that are viable were detected. Subsequently, RNA was extracted from lysed cells, selectively amplified using NASBA, and rapidly identified by the biosensor. While the biosensor assay requires only 15 min assay time, the overall process takes 4 h for detection of ten viable B. anthracis spores, and is shortened significantly if more spores are present. The biosensor is based on an oligonucleotide sandwich-hybridization assay format. It uses a membrane flow-through system with an immobilized DNA probe that hybridizes with the target sequence. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye sulforhodamine B. The amount of liposomes captured in the detection zone can be read visually or quantified with a hand-held reflectometer. The biosensor can detect as little as 1 fmol target mRNA (1 nmol L^–1). Specificity analysis revealed no cross-reactivity with 11 organisms tested, among them closely related species such as B. cereus, B. megaterium, B. subtilis, B. thuringiensis, Lactococcus lactis, Lactobacillus plantarum, and Chlostridium butyricum. Also, no false positive signals were obtained from nonviable spores. We suggest that this inexpensive biosensor is a viable option for rapid, on-site analysis providing highly specific data on the presence of viable B. anthracis spores.     

Trypsin and MALDI matrix pre‐coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry

Prefabricated surfaces containing α‐cyano‐4‐hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α‐cyano‐4‐hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography‐tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine‐rich C‐kinase substrate (29.8 kDa) and spectrin alpha chain, non‐erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre‐coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. Copyright © 2016 John Wiley & Sons, Ltd.     

Fast trypsin digestion of proteins on a cross‐linked [Os(dmebpy)2Cl]+/2+‐derivatized copolymer of acrylamide and vinylimidazole column

Fast digestion of proteins was observed when they were loaded together with trypsin onto the cross‐linked [Os(dmebpy)₂Cl]^+/2+‐derivatized copolymer of acrylamide and vinylimidazole column. The insoluble Os‐complexed polymer particles were packed into an electrospray tip to monitor peptides eluted during loading, washing and elution periods with a mass spectrometer. The proteolytic cleavage of proteins was observed immediately when the mixture of trypsin and substrates in 0.2 mM ammonium bicarbonate 50:50 H₂O/acetonitrile reached the column tip, and continued through the loading period. Some tryptic peptides were released from the column during the loading and following washing periods. The others still stayed on the column until the low pH elution buffer reached the column. If a protein was first loaded onto the column, no tryptic peptides of the protein were observed when trypsin was loaded later for the on‐column digestion. Only the autolysis peptides of trypsin were observed. On‐column digestion of 100 fmol myoglobin was successfully detected with a low sensitivity quadrupole mass spectrometer. A hybrid Os‐polymer/C₁₈ column tip was constructed for the online trypsin digestion of proteins in the aqueous buffers and the following trapping and elution of peptides from the C₁₈ column. The digestion of reduced and alkylated bovine serum albumin and human transferrin in 2.5 mM ammonium bicarbonate and 0.2 M urea buffer was observed on the column, with more peptide coverage than conventional 4 h in‐solution digestion at 37°C. Control experiments without the Os‐polymer in the column tip excluded the spontaneous in‐solution digestion of proteins in the short time window of buffer delivery onto the column, indirectly confirming the contribution of Os‐polymer on the fast trypsin digestion. Copyright © 2010 John Wiley & Sons, Ltd.     

Improvement of gel‐separated protein identification by DMF‐assisted digestion and peptide recovery after electroblotting

In‐gel digestion of gel‐separated proteins is a major route to assist in proteomics‐based biological discovery, which, however, is often embarrassed by its inherent limitations such as the low digestion efficiency and the low recovery of proteolytic peptides. For overcoming these limitations, many efforts have been directed at developing alternative methods to avoid the in‐digestion. Here, we present a new method for efficient protein digestion and tryptic peptide recovery, which involved electroblotting gel‐separated proteins onto a PVDF membrane, excising the PVDF bands containing protein of interest, and dissolving the bands with pure DMF (≥99.8%). Before tryptic digestion, NH₄HCO₃ buffer was added to moderately adjust the DMF concentration (to 40%) in order for trypsin to exert its activity. Experimental results using protein standards showed that, due to actions of DMF in dissolving PVDF membrane and the membrane‐bound substances, the proteins were virtually in‐solution digested in DMF‐containing buffer. This protocol allowed more efficient digestion and peptide recovery, thereby increasing the sequence coverage and the confidence of protein identification. The comparative study using rat hippocampal membrane‐enriched sample showed that the method was superior to the reported on‐membrane tryptic digestion for further protein identification, including low abundant and/or highly hydrophobic membrane proteins.     

Detection and identification of arginine modifications on methylglyoxal-modified ribonuclease by mass spectrometric analysis

Analysis of the broad range of trace chemical modifications of proteins in biological samples is a significant challenge for modern mass spectrometry. Modification at lysine and arginine residues, in particular, causes resistance to digestion by trypsin, producing large tryptic peptides that are not readily sequenced by mass spectrometry. In this work, we describe the analysis of ribonuclease (RNase) modified by methylglyoxal (MGO) under physiological conditions. For detection of modifications, we use comparative analysis of the single combined spectra extracted from the full-scan MS data of the tryptic digests from native and modified proteins. This approach revealed 11 ions unique to MGO-modified RNase, including a 32-amino acid peptide containing a modified Arg-85 residue. Sequential digestion of MGO-modified RNase by endoproteinase Glu-C and trypsin was required to obtain peptides that were amenable to sequencing analysis. Arg-39 was identified as the main site of modification (35% modification) on MGO-modified Rnase, and the dihydroxyimidazolidine and hydroimidazolone derivatives were the main adducts formed, with minor amounts of the tetrahydropyrimidine and argpyrimidine derivatives. For identification of these products, we used variations in source voltage and collision energy to obtain the dehydration and decarboxylation products of the tetrahydropyrimidine-containing peptides and dehydration of the dihydroxyimidazoline-containing peptides. The resultant spectra were dependent on the cone voltage and collision energy, and analysis of spectra at various settings permitted structural assignments. These studies illustrate the usefulness of single combined mass spectra extracted from full-scan data and variations in source and collision cell voltages for detection and structural characterization of chemical adducts on proteins.     

Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin

Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin‐IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin‐ IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.     

Selection of possible marker peptides for the detection of major ruminant milk proteins in food by liquid chromatography-tandem mass spectrometry

The aim of this work was the determination of peptides, which can function as markers for identification of milk allergens in food samples. Emphasis was placed on two casein proteins (α- and β-casein) and two whey proteins (α-lactalbumin and β-lactoglobulin). In silico tryptic digestion provided preliminary information about the expected peptides. After tryptic digestion of four milk allergens, the analytical data obtained by combination of reversed-phase high performance liquid chromatography and quadrupole tandem mass spectrometry (LC-MS/MS) led to the identification of 26 peptides. Seven of these peptides were synthesized and used for calibration of the LC-MS/MS system. Species specificity of the selected peptides was sought by BLAST search. Among the selected peptides, only LIVTQTMK from β-lactoglobulin (m/z 467.6, charge 2+) was found to be cow milk specific and could function as a marker. Two other peptides, FFVAPFPEVFGK from α-casein (m/z 693.3, charge 2+) and GPFPIIV from β-casein (m/z 742.5, charge 1+), occur in water buffalo milk too. The other four peptides appear in the milk of other species also and can be used as markers for ruminant species milk. Using these seven peptides, a multianalyte MS-based method was developed. For the establishment of the method, it was applied at first to different dairy samples, and then to chocolate and blank samples, and the peptides could be determined down to 1 ng/mL in food samples. At the end, spiked samples were measured, where the target peptides could be detected with a high recovery (over 50%).     

Protein identification by accurate mass matrix-assisted laser desorption/ionization imaging of tryptic peptides

The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix‐assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on‐tissue identification of tryptic peptides by accurate mass measurements and complementary off‐line liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) analysis. Two adjacent mouse brain sections were analyzed in parallel. The first section was spotted with trypsin and analyzed by MALDI imaging. Direct on‐tissue MS/MS experiments of this section resulted in the identification of 14 peptides (originating from 4 proteins). The second tissue section was homogenized, fractionated by ultracentrifugation and digested with trypsin prior to LC/ESI‐MS/MS analysis. The number of identified peptides was increased to 153 (corresponding to 106 proteins) by matching imaged mass peaks to peptides which were identified in these LC/ESI‐MS/MS experiments. All results (including MALDI imaging data) were based on accurate mass measurements (RMS <2 ppm) and allow a confident identification of tryptic peptides. Measurements based on lower accuracy would have led to ambiguous or misleading results. MS images of identified peptides were generated with a bin width (mass range used for image generation) of Δm/z = 0.01. The application of accurate mass measurements and additional LC/MS measurements increased both the quality and the number of peptide identifications. The advantages of this approach for the analysis of biological tissue sections are demonstrated and discussed in detail. Results indicate that accurate mass measurements are needed for confident identification and specific image generation of tryptic peptides in tissue sections. Copyright © 2011 John Wiley & Sons, Ltd.     

Detergent‐assisted sample preparation for MALDI‐MS: Investigation of octylglucoside and docecylmaltoside for matrix crystallization,on‐plate digestion,and trypsin activity

We show an easy and fast method for improved detection of lipophilic peptides with MALDI‐MS utilizing the nonionic detergents n‐octylglucoside and n‐dodecylmaltoside (laurylmaltoside). Investigations comprised on‐plate digestion of proteins with trypsin, detergent effects on the protease trypsin, and the changes in MALDI matrix crystallization. Investigations also exhibited a higher tryptic activity in trypsin activity assay of 139% when using laurylmaltoside as supplement. Crystallization changed toward a more homogeneous crystal distribution and especially trypsinized insulin spectra recorded with MALDI‐MS showed improved detectability of lipophilic peptides.     

On particle ionization/enrichment of multifunctional nanoprobes: washing/separation-free, acceleration and enrichment of microwave-assisted tryptic digestion of proteins via bare TiO2 nanoparticles in ESI-MS and comparing to MALDI-MS

A simple, rapid, straightforward and washing/separation free of in-solution digestion method for microwave-assisted tryptic digestion of proteins (cytochrome c, lysozyme and myoglobin) using bare TiO(2) nanoparticles (NPs) prepared in aqueous solution to serve as multifunctional nanoprobes in electrospray ionization mass spectrometry (ESI-MS) was demonstrated. The current approach is termed as 'on particle ionization/enrichment (OPIE)' and it can be applied in ESI-MS, atmospheric pressure-matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The bare TiO(2) NPs can assist, accelerate and effectively enhance the digestion efficiency, sequence coverage and detection sensitivity of peptides for the microwave-assisted tryptic digestion of proteins in ESI-MS. The reason is attributed to the fact that proteins or partially digested proteins are easily attracted or concentrated onto the surface of TiO(2) NPs, resulting in higher efficiency of digestion reactions in the microwave experiments. Besides, the TiO(2) NPs could act as a microwave absorber to accelerate and enrich the protein fragments in a short period of time (40-60 s) from the microwave experiments in ESI-MS. Furthermore, the bare TiO(2) NPs prepared in aqueous solution exhibit high adsorption capability toward the protein fragments (peptides); thus, the OPIE approach for detecting the digested protein fragments via ESI and MALDI ionization could be achieved. The current technique is also a washing and separation-free technique for accelerating and enriching microwave-assisted tryptic digestion of proteins in the ESI-MS and MALDI-MS. It exhibits potential to be widely applied to biotechnology and proteome research in the near future.     

Integrating Lys-N proteolysis and N-terminal guanidination for improved fragmentation and relative quantification of singly-charged ions

Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA The study of isolated protein complexes has greatly benefited from recent advances in mass spectrometry instrumentation and quantitative, isotope labeling techniques. The comprehensive characterization of protein complex components and quantification of their relative abundance relies heavily upon maximizing protein and peptide sequence information obtained from MS and tandem MS studies. Recent work has shown that using a metalloendopeptidase, Lys-N, for proteomic analysis of biological protein mixtures produces complementary protein sequence information compared with trypsin digestion alone. Here, we have investigated the suitability of Lys-N proteolysis for use with MALDI mass spectrometry to characterize the yeast Arp2 complex and E. coli PAP I protein interactions. Although Lys-N digestion resulted in an average decrease in protein sequence coverage of ∼30% compared with trypsin digestion, CID analysis of singly-charged Lys-N peptides yielded a more extensive b-ions series compared with complementary tryptic peptides. Taking advantage of this improved fragmentation pattern, we utilized differential ¹⁵N/¹⁴N guanidination of Lys-N peptides and MALDI-MS/MS analysis to relatively quantify the changes in PAP I associations due to deletion of sprE, previously shown to regulate PAP I-dependent polyadenylation. Overall, this Lys-N/guanidination integrative approach is applicable for functional proteomic studies utilizing MALDI mass spectrometry analysis, as it provides an effective and economical mean for relative quantification of proteins in conjunction with increased sensitivity of detection and fragmentation efficiency.     

Investigations on the Interactions of λPhage-Derived Peptides Against the SrtA Mechanism in Bacillus anthracis

Bacillus anthracis is a well-known bioweapon pathogen, which coordinates the expression of its virulence factors in response to a specific environmental signal by its protein architecture. Absences of sortase signal functioning may fail to assemble the surface linked proteins and so B. anthracis cannot sustain an infection with host cells. Targeting the signaling mechanism of B. anthracis can be achieved by inhibition of SrtA enzyme through λphage-derived plyG. The lysin enzyme plyG is experimentally proven as bacteriolytic agent, specifically kill's B. anthracis by inhibiting the SrtA. Here, we have screened the peptides from λphage lysin, and these peptides are having the ability as LPXTG competitive inhibitors. In comparison to the activator peptide LPXTG binding motif, λphage lysin based inhibitor peptides are having much supremacy towards binding of SrtA. Finally, peptide structures extracted from PlyG are free from toxic, allergic abilities and also have the ability to terminate the signal transduction mechanism in B. anthracis.     

Primary structure of β-momorcharin,a ribosome-inactivating protein from the seeds of Momordica Charantia Linn (Cucurbitaceae)

The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S.aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 51 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.     

Rapid characterization of Bacillus spores targeting species-unique peptides produced with an atmospheric pressure matrix-assisted laser desorption/ionization source

New and improved strategies are eagerly sought for the rapid identification of microorganisms, particularly in mixtures. Mass spectrometry remains a powerful tool for this purpose. Small acid-soluble proteins (SASPs), which are relatively abundant in Bacillus spores, represent potential biomarkers for species characterization. Despite sharing extensive sequence homology, these proteins differ sufficiently in sequence for discrimination between species. This work focuses on the differences in sequence between SASPs from various Bacillus species. Compilation of SASP sequences from protein database searches, followed by in silico trypsin digestion and analysis of the resulting fragments, identified several species-specific peptides that could be targeted for analysis using mass spectrometry. This strategy was tested and found to be successful in the characterization of Bacillus spores both from individual species and in mixtures. Analysis was performed using an ion trap mass spectrometer with an atmospheric pressure MALDI source. This instrumentation offers the advantage of increased speed of analysis and accurate precursor ion selection for tandem mass spectrometric analysis compared with vacuum matrix-assisted laser desorption/ionization and time-of-flight instruments. The identification and targeting of species-specific peptides using this type of instrumentation offers a rapid, efficient strategy for the identification of Bacillus spores and can potentially be applied to different microorganisms.     

Bacillus anthracis: toxicology, epidemiology and current rapid-detection methods

B. anthracis, the causative agent for anthrax, has been well studied for over 150 years. Due to the genetic similarities among various Bacillus species, as well as its existence in both a spore form and a vegetative state, the detection and specific identification of B. anthracis have been proven to require complex techniques and/or laborious methods. With the heightened interest in the organism as a potential biological threat agent, a large number of interesting detection technologies have recently been developed, including methods involving immunological and nucleic acid-based assay formats. The technologies range from culture-based methods to portable Total Analysis Systems based on real-time PCR. This review with 170 references provides a brief background on the toxicology and epidemiology of B. anthracis, discusses challenges associated with its detection related to genetic similarities to other species, and reviews immunological and, with greater emphasis, nucleic acid-based detection systems. Harriet A. Clancy is currently on active duty with the U.S. Army’s 3rd COSCOM (Corps Support Command)     

Vortex-assisted tryptic digestion

The effect of vortex‐induced vibration during tryptic digestion was investigated by applying different vibrational speeds (0, 600, 1200, or 2500 rpm) to digestion solutions for varying durations (10, 20, 30, 40, or 60 min) at two different incubation temperatures (25°C or 37°C). The most rapid digestion was observed with the highest vibrational speed and temperature. With the application of 2500 rpm at 37°C, the tryptic digestion of each of three standard proteins (cytochrome c, myoglobin, or bovine serum albumin) provided complete disappearance of the protein within 60 min, as determined by matrix‐assisted laser desorption/ionization mass spectrometry. Compared to conventional overnight digestion, 60‐min vortex‐assisted tryptic digestion generated longer peptides, due primarily to the limited digestion time and provided better sequence coverages (89% vs. 78% for cytochrome c, 100% vs. 87% for myoglobin, and 38% vs. 26% for BSA). The longer peptides should be advantageous to analytical methods such as the middle‐down approach that benefit from increased sequence coverage of proteins. Vortex‐assisted tryptic digestion is expected to be a useful method for rapid tryptic digestion of proteins. Copyright © 2010 John Wiley & Sons, Ltd.     

Sample preparation by in-gel digestion for mass spectrometry-based proteomics

The proteomic characterization of proteins and protein complexes from cells and cell organelles is the next challenge for investigation of the cell. After isolation of the cell compartment, three steps have to be performed in the laboratory to yield information about the proteins present. The protein mixtures must be separated into single species, broken down into peptides, and, finally, identified by mass spectrometry. Most scientists engaged in proteomics separate proteins by electrophoresis. For characterization and identification of proteomes, mass spectrometry of peptides is the method of choice. To combine electrophoresis and mass spectrometry, sample preparation by “in-gel digestion” has been developed. Many procedures are available for in-gel digestion, which inspired us to review in-gel digestion approaches. Figure Classical in-gel digestion process for a protein band stained with CBB. Protein bands are cut from the polyacrylamide gel (1). CBB molecules (blue circles) bound to the protein are released by iterative incubation in a buffered organic solvent system (2). To increase digestion efficiency and sequence coverage proteins are reduced (3) and alkylated (4). Proteins are subsequently digested with proteolytic enzymes (scissors symbols), typically trypsin (5). Trypsin cleaves at the amino acid residues arginine (R) and lysine (K). The resulting peptides (A, B, and C) are extracted from the polyacrylamide matrix (6). The peptide solution can be further purified for analysis by mass spectrometry (Section “Concentration and desalting of peptides”)     

Molecular mass spectrometric identification of superoxide dismutase in the liver of mice <Emphasis Type="Italic">Mus musculus</Emphasis> and <Emphasis Type="Italic">Mus spretus</Emphasis> using a metallomics analytical approach

This paper reports the identification and quantification of superoxide dismutase in the liver of Mus musculus and Mus spretus mice using a metallomics analytical approach. The approach consisted of using orthogonal chromatographic systems coupled to ICP–MS and UV detectors. Size-exclusion fractionation of the cytosolic extracts was followed by anion-exchange chromatographic separation of Cu- and Zn-containing species. After purification then tryptic digestion, Cu- and Zn-containing superoxide dismutase was identified by nESI-QqTOF. The MS–MS spectra of doubly charged peptides, with the Mascot searching engine, were used to obtain the sequence of the protein.     

Integration of capillary isoelectric focusing with monolithic immobilized pH gradient,immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis

An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M‐IPG), on‐line digestion by trypsin‐based immobilized enzyme microreactor (trypsin‐IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M‐IPG CIEF column and trypsin‐IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20 mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M‐IPG CIEF, followed by on‐line digestion by trypsin‐IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE‐based on‐line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.     

Differentiation of strains from the Bacillus cereus group by RFLP‐PFGE genomic fingerprinting

Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus anthracis, Bacillus thuringiensis, and Bacillus cereus belong to the B. cereus group. The last three species are characterized by different phenotype features and pathogenicity spectrum, but it has been shown that these species are genetically closely related. The macrorestriction analysis of the genomic DNA with the NotI enzyme was used to generate polymorphism of restriction profiles for 39 food‐borne isolates (B. cereus, B. mycoides) and seven reference strains (B. mycoides, B. thuringiensis, B. weihenstephanensis, and B. cereus). The PFGE method was applied to differentiate the examined strains of the B. cereus group. On the basis of the unweighted pair group method with the arithmetic mean method and Dice coefficient, the strains were divided into five clusters (types A–E), and the most numerous group was group A (25 strains). A total of 21 distinct pulsotypes were observed. The RFLP‐PFGE analysis was successfully used for the differentiation and characterization of B. cereus and B. mycoides strains isolated from different food products.