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Submitted to: Journal of Separation Science
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 3/1/2012 Publication Date: 3/2/2012 Citation: Gunther, N.W., Paul, M., Nunez, A., Liu, Y. 2012. pH fractionation and identification of proteins: comparing column isoelectric focusing vs liquid based focusing techniques. Journal of Separation Science. 35(12):1399-1406. Interpretive Summary: To understand how food-borne pathogens survive and grow in food, it is necessary to be able to identify the proteins, which the bacteria produce that enable their persistence. However, many bacteria are capable of producing >4000 proteins at any given time. Therefore it is helpful in this field of research to be able to subdivide the proteins produced by the bacteria into smaller more manageable groups for further investigation. In this work we compared two separation techniques that use similar methods for fractioning all of the proteins produced by a sample of bacteria into smaller well defined groups for easier analyses. We found that the proteins in the subgroups produced by these two methods were distinctly different but not immediately superior to one another. However, concerns about the costs and decreased flexibility inherent in using one of the separation techniques led to the other technique being determined to be a more practical approach for this type of bacterial food safety research. The information obtained from this research will allow us to more easily identify proteins that bacteria utilize to survive and grow in food and will aid in the design of novel control strategies targeting the bacteria’s metabolic pathways that are essential for their successful contamination of the food supply. Technical Abstract: In order to understand how bacteria react and adapt to changes in the environment, it is necessary to identify the proteins the bacteria produces under different environmental conditions. However, the proteomes of even simple organisms like bacteria can contain a significant number of proteins, making the identification of all proteins in a proteome in every instance costly in both time and resources. Historically, different separation methods have been developed and utilized to fractionate whole proteomes into more manageable discrete fractions. In this work, using a quadrupole time of flight mass spectrometer (Q-TOF) as the technology for identifying proteins, two different isoelectric focusing (IEF) fractionation methods were compared and contrasted to determine the potential of each method for the simple and reproducible fractionation of a whole bacterial proteome. Isoelectic focusing using a column based approach and a liquid based technique for fractionation of the proteome based on the isoelectric point (pI) of the proteins produced similar results in terms of overall sensitivity (378 and 409 total proteins) and reproducibility (less than 10% change) in the proteins identified. However, there was limited overlap in the protein sets with less then 50% of the total proteins identified being common to both sets of proteins identified by the two different system. In addition to the sensitivity and reproducibility of data derived from the two methods; overall cost and flexibility of each of these techniques was considered in order to adopt a system best suited for comparative proteomic projects. |
