Bacterial characterization using protein profiling in a microchip separations platform

Abstract

A rapid microanalytical protein-based approach to bacterial characterization is presented. Chip gel electrophoresis (CGE) coupled with LIF detection was used to analyze lysates from different bacterial cell lines to obtain signature profiles of the soluble protein composition. The study includes Escherichia coli, Bacillus subtilis, and Bacillus anthracis (Delta Sterne strain) vegetative cells as well as endospores formed from the latter two species as model organisms to demonstrate the method. A unified protein preparation protocol was developed for both cell types to streamline the benchtop process and aid future automation. Cells and spores were lysed and proteins solubilized using a combination of thermal and chemical lysis methods. Reducing agents, necessary to solubilize spore proteins, were eliminated using a small-scale rapid size-exclusion chromatography step to eliminate interference with down-stream protein labeling. This approach was found to be compatible with nonspore cells (i.e., vegetative cells) as well, not adversely impacting the protein signatures. Data are presented demonstrating distinct CGE protein signatures for our model organisms, suggesting the potential for discrimination of organisms on the basis of empirical protein patterns. The goal of this work is to develop a fast and field-portable method for characterizing bacteria via their proteomes.

Separation of similar yeast strains by IEF techniques

“…They are laborious and expensive and they cannot detect phenotypic expression of the biofilm formation in yeasts. Similarly, protein and peptide patterns are being increasingly mentioned in the literature related to microbial identification [9]. MALDI-TOF-MS is a useful technique for the identification of whole or lysed microorganisms (MOs) on the basis of their characteristic protein mass spectrum fingerprint [10][11][12] or on the proteomic microbial identification [13,14], respectively.…”

Development of an integrated microfluidic instrument for unattended water‐monitoring applications

“…Organism detection will require additional work on sample preparation and signature detection. The basic capabilities have already been developed under other funding [10,11]; additional modular components to concentrate organisms, lyse them and solubilize their proteins can be readily incorporated into the existing microfluidic architecture of the UWS. If widely implemented in municipal water utilities throughout the country, a technology like the UWS could provide significant cost savings to the utilities and their customers.…”

“…The chip itself is mounted in a liquid module similar to what has been previously described [8][9][10][11]. Modifications of the previous design were made to facilitate extended periods of unattended operation, including a larger buffer reservoir to delay the effects of buffer depletion and overflow ports on the reservoirs leading to a collection vessel to prevent possible damage to the system from extended operations.…”

Profil Protein Bakteri Ureolitik

“…Protein profiling bertujuan untuk melihat protein yang diturunkan dari organisme untuk menghasilkan informasi tentang protein organisme secara keseluruhan [10]. Marka potein dapat digunakan untuk mengidentifikasi berat molekul dari campuran polipeptida.…”