Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.
The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.
The genus Burkholderia comprises more than 62 species, Gram negative organisms isolated from a wide range of niches, and it is divided in two main clusters1,2. The first cluster includes human, animal and phytotrophic organisms, and most studies have focused on the pathogenic species of this group due to their clinical importance. The most pathogenic members are B. pseudomallei and B. mallei (which causes melioidosis and glanders respectively)3,4 and opportunistic pathogens (the 17 defined species of the Burkholderia cepacia complex, BCC)5, which cause disease in cystic fibrosis (CF) and chronic granulomatous disease (CGD)1. The second cluster, with more than 30 nonpathogenic species, includes bacteria associated with plants or with the environment, and are considered potentially beneficial to the host2.
Numerous complications emerge from bacterial infection with the pathogenic members of the Burkholderia genus, such as transmission of the pathogen between patients, spread of the disease and treatment failure because of the intrinsic or acquired resistance to antibiotics making hard to eradicate in most of the cases6-9. Therefore, gaining a clearer understanding of the basis for establishment of bacterial infection is vital to the treatment of diseases caused by these organisms. In order to gain insight into the establishment of infection, extensive investigations on the bacterial components associated with pathogenesis are needed. Studies focusing on the proteomic analysis of Burkholderia organisms using proteomic approaches described proteins that have been implicated in bacterial pathogenesis as well as changes in their proteome profiles10-16.
Protein extraction methods using sonication and freeze-thawed cycles in lysis buffer containing high concentration of urea, thiourea in combination with detergent and ampholytes has been applied in Burkholderia proteomic studies10-13. Although urea is quite efficient for protein denaturation, it can establish an equilibrium in aqueous solution with ammonium isocyanate, which can react with amino acid groups, thereby forming artifacts (carbamylation reaction)17. Therefore, it is recommended to include carrier ampholytes, which act as cyanate scavengers and avoid temperatures above 37 °C17. Furthermore, to prevent any chemical interference of lysis buffer with protein quantification, the same lysis buffer can be used to generate the standard curve so that the samples and the standards have the same background10. Other methodologies involve the use of alkaline buffers and detergents with heat incubation periods17,18; however these conditions might induce changes in the proteome and some detergents are not compatible with proteomics application unless subsequent detergent removal steps are included17,18.
After adequate extraction and quantification, global protein expression of each individual protein can be studied using proteomic approaches such as two-dimensional (2-D) gel electrophoresis. This technique was first described by O'Farell19 and consists in the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, and then according to their molecular weight by acrylamide gel electrophoresis in the second dimension. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources19,20. This separation technique is currently available in protein-centric approaches with the great advantage of resolving protein isoforms caused by post-transcriptional modifications or proteolytic processing. Quantitative changes can be detected by comparing the intensity of the corresponding spot after staining of the gel20. However, this technique is not suited for the identification of very large proteins, membrane proteins, extremely basic and acidic or hydrophobic proteins, and is a somewhat laborious and time-consuming technique20. New peptide-centric approaches (non gel-based) that are more robust and objective become available and can be used for quantitative comparison by differential stable isotope labeling methods such as cysteine labeling by isotope-coded affinity tagging (ICAT)21, and amino group labeling by isotope tagging for relative and absolute quantitation (iTRAQ)22. The use of a single proteomic technique might give insufficient information; therefore the use of two complementary proteomic approaches is necessary for most fully assessing changes in proteome. Nevertheless, 2-D gel electrophoresis is widely used and can be routinely applied for quantitative expression of several proteins in different organisms.
Here we describe a whole-cell protein extraction and 2-D gel electrophoresis procedures for Burkholderia species that were adapted and optimized from the GE Healthcare 2-D Electrophoresis Principles and methods Handbook 80-6429-60AC 2004 (www.amershambiosciences.com). The protein extraction was carried out using a bead beater with glass beads in the presence of PBS containing 5 mM EDTA (ethylenediamine tetraacetic acid) and 1 mM PMSF (phenylmethylsulfonyl fluoride). This procedure allows quantification of proteins with minimal degradation and is amendable for proteomic approaches as previously reported15,23,24. 2-D gel electrophoresis was carried out using 24 cm long immobilized pH 4-7 gradient and proteins were separated according to their isoelectric point. Then proteins were separated according to their molecular weight by SDS-polyacrylamide gel. Additionally, we described a silver staining method for visualization of spot proteins, and a silver stain method that is compatible for mass spectrometry analysis. Together these procedures can allow the identification of important proteins from Burkholderia species that could be involved in pathogenesis.
1. Culture Growth (Days 1+2)
2. Protein Extraction (Day 3)
3. Sample Preparation (Day 4)
4. First-dimension Isoelectric Focusing (IEF) (Day 4)
All steps in this section are using the Ettan IGPhor IEF System.
5. Second-dimension SDS-polyacrylamide Gel Electrophoresis (Day 5)
All steps in this section are using the Ettan DALTsix electrophoresis apparatus.
6. Silver Staining of the Gels for Gel Visualization (Day 5-6)
7. Gel Silver Staining for Mass Spectrometry Analysis
Comparative analysis of the protein profiles extracted from the same bacterial culture on two different occasions showed similar patterns banding indicating successful protein extractions. Molecular weight proteins extracted ranged from 10-150 kDa. Figure 1 shows representative Coomassie blue staining gel of the whole-cell protein extractions from Burkholderia multivorans (a member of the BCC) clinical isolates grown in LB or Yeast/Manitol (YEM) broth and harvested from stationary phase.
For 2-D gel electrophoresis analysis, 200 μg of total proteins from Burkholderia pseudomallei was used (Figure 2). Since this pathogen is recognized as a B-type biological warfare agent by the US Centers for Disease Control and Prevention25, protein preparation procedures were carried out in Bio Safety Level 3 containment laboratory and accordingly with standard operation procedures. Proteins were resuspended in rehydration solution and applied to a 24 cm long immobilized pH 4-7 gradient and proteins were separated according to their isoelectric point (pI). Then strips were placed on a SDS-polyacrylamide gel and proteins were separated according to their molecular weight (MW). Subsequently, gel was silver stained for the protein spot visualization. Results showed the abundance of more than 500 protein spots that can be individually identified by mass spectrometry.
Figure 1. Total protein profiles of Burkholderia multivorans isolates. SDS-PAGE analysis of protein extracted at the stationary phase from D-2214 and D-2094 isolates grown in LB or Yeast/Manitol (YEM) broth in two different occasions (1 and 2). 4 μg of protein were loaded in each lane of a 12.5% gel and stained with Coomassie blue. Lane M, protein marker.
Figure 2. 2-D gel electrophoresis analysis of Burkholderia pseudomallei 1234B isolate. Representative silver-stained 2-D gel showing protein separation in the pI 4 to 7-range strip. Whole-cell protein extracts (200 μg) at the stationary phase were used and separated on a 15% SDS-PAGE gel.
A method for proteins preparation has been described that can extract the majority of Burkholderia proteins with good reproducibility. This is demonstrated by obtaining the same protein profile from two independent preparations performed in different days using the same bacterial culture grown in LB or YEM broth as shown in Figure 1. Extraction was efficient for bacteria grown in liquid media; however we have not tested this method for bacteria grown on plates. This method was also used for further protein characterization using proteomic tools; our group has successfully studied proteome changes using 2-D gel electrophoresis and isotope tagging for relative and absolute quantitation (iTRAQ) proteomic techniques15,23,24. For the latter, proteins were extracted into a buffer containing 0.5 M EDTA in PBS to avoid any interference of PMSF with the iTRAQ experiments15.
It is important to remark that during the protein extraction process and 2-D gel electrophoresis, all steps should be carried out at 4 °C or in cold conditions using ice buckets to minimize protein degradation, as well as to use plugged tips and wear nitrile gloves and cover all skin to prevent keratin contamination of any spots cut out for subsequent identification by mass spectrometry. When performing protein extractions, it is also important to consider that the PMSF has a short half-life time in aqueous solutions, according to the manufacturer, therefore the 0.1 mM stock solution in acetone should be made immediately before use. Alternatively, other serine inhibitors or protease inhibitors that are more soluble and stable, and less toxic can be used, although we have not tested them in our experiments.
The lysis buffer used in this procedure does not contain urea, which is important for the solubilization to extract both aqueous (cytosolic) proteins and less soluble proteins, including some membrane proteins17. But, during sample preparation for first-dimension isoelectric focusing (step 3 of this protocol), samples are resuspended in rehydration buffer that contains urea. Other proteomic techniques also involve similar treatment26. Before following with the rehydration step, it seems that the removal of interfering materials or low molecular weight impurities is crucial for high resolution, we recommend to use the 2-D Clean-up Kit in each protein samples as results improved. Previously loading the samples for first-dimension isoelectric focusing separation, make sure to have prepared strips of blotting paper the right size of the strip holders, as this is problematic to cut the right size in aseptic fashion on the spot (for step 3.4 of this protocol).
Before running a 2-D SDS-polyacrylamide gel electrophoresis, it is necessary to ensure that the gel percentage fits the expected sizes of interest (a 12.5% percentage gel of can resolve protein in the range of 14-100 kDa). After second dimension gel electrophoresis is completed, visualization of proteins can be achieved by Coomassie blue or silver staining; the latter staining is more sensitive with detection limit is as low as 0.1 ng/protein/spot20. Protein spots visualized with silver staining are suitable for further analysis by mass spectrometry. However, it is important to mention that silver stain solution should not contain glutaraldehyde since this agent crosslinks with proteins; therefore it is not compatible with further mass spectrometry analysis20. Alternatively, to detect and quantify differentially expressed proteins, another gel-based approach such as Two Dimensional-Difference In Gel Electrophoresis (2D-DIGE) together with automated software can be used. This approach employs distinct fluorescent tags (e.g. Cy 3, 5 and 2) that are used to label samples and a universal internal standard prior to second dimension electrophoresis overcoming, to some degree, the disadvantages of variation and reproducibility of 2-D electrophoresis27. Additionally, gels can be stained after the second dimension with SyproRuby, which is an organometallic ruthenium chelate stain designed for proteomic applications. It can detect protein spots with similar sensitivity to that silver staining, but with greater sensitivity than Coomassie blue. After staining gels can be photographed with laser scanner or transilluminator28.
In conclusion, this video describes a whole-cell bacterial protein extraction procedure and 2-D gel electrophoresis method that can allow the study of global changes in the proteome in Burkholderia species.
The authors have nothing to disclose.
This work was supported by a studentship from The University of British Columbia (to B.V.) and grants from Cystic Fibrosis Canada and Canadian Institutes of Health Research (to D.P.S.). We thank Jacqueline Chung for the initial preparation of protocols.
Reagents | |||
Phenylmethanesulfonyl fluoride (PMSF) | Sigma | P7626 | Toxic, corrosive |
Acetone | Fisher | A18-1 | Flammable |
PBS Buffer | Bioscience | R028 | |
Ethylenediaminetetraacetic acid (EDTA) | Fisher | BP118-500 | toxic |
Glass beads (0.1 mm) | BioSpec Products | 11079101 | |
Nalgene Oak Ridge Centrifuge tubes, polycarbonate (50 ml) | VWR | 21009-342 | |
MicroBCA protein extraction kit | Pierce | 23235 | |
Microcentrifuge Tubes 2.0 ml conical Screw cap Tubes with Cap and O-Ring | Fisher | 02-681-375 | |
2-D Clean-Up kit | GE Healthcare | 80-6484-51 | |
Urea | Invitrogen | 15505-050 | Irritant |
CHAPS | Amersham Biosciences | 17-1314-01 | |
Dithiothreitol (DTT) | MPBiomedical, LCC | 856126 | Irritant |
Immobiline DryStrips pH 4-7 24 cm | Amersham Biosciences | 176002-46 | |
Duracryl | Proteomic Solutions | 80-0148 | Very toxic, carcinogen |
Ammonium persulfate | Fisher | BP179-100 | Flammable, toxic, corrosive |
N,N,N’,N’-tetramethylethylenediamine (TEMED) | Invitrogen | 15524-010 | Flammable |
Sodium dodecyl sulfate (SDS) | Fisher | BP166-500 | Acute toxicity, flammable |
Agarose | Invitrogen | 15510-027 | |
Ethanol | Fisher | HC1100-1GL | Flammable, toxic |
Acetic acid | Fisher | A491-212 | Flammable, corrosive |
Glutaraldehyde | Fisher | G151-1 | Very toxic, corrosive, dangerous for the environment |
Potassium tetrathionate | Sigma | P2926 | Irritant |
Sodium acetate | EM Science | 7510 | |
Silver nitrate | Sigma | 209139 | Corrosive, dangerous for the environment |
Formaldehyde | Sigma | 252549 | Toxic |
Sodium thiosulfate | Sigma | S7026 | |
Tris Base | EMD | 9230 | |
Glycine | MPBiomedical, LCC | 808831 | |
Glycerol | MPBiomedical, LCC | 800688 | |
Methanol | Fisher | A412-4 | Flammable, toxic, health hazard |
Sodium carbonate anhydrous | EMD | SX0400-3 | Toxic |
Mineral oil | ACROS | 415080010 | |
Equipment | |||
JA-20 ultracentrifuge rotor | Beckman Coulter | 334831 | |
Mini Beadbeater | Biospec Products | 3110BX | |
Ettan IPGphor II Isoelectric Focusing System and accessories | GE Healthcare | 80-6505-03 | www.amershambiosciences.com |
Ettan DALT Large Vertical electrophoresis system | GE Healthcare | 80-6485-27 | www.amershambiosciences.com |