Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

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 clusters^1,2. 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 cases^6-9. 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 profiles^10-16.

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 studies^10-13. 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 periods^17,18; however these conditions might induce changes in the proteome and some detergents are not compatible with proteomics application unless subsequent detergent removal steps are included^17,18.

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 sources^19,20. 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 reported^15,23,24. 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.