J. Sep. Sci. 2012, 35, 1399–1405 1399 Nereus W. Gunther IV 1* Moushumi Paul 2* Alberto Nu ˜ nez 3* Yanhong Liu 1 1 Molecular Characterization of Foodborne Pathogens Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA, USA 2 Dairy and Functional Foods Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA, USA 3 Core Technologies, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA, USA Received September 30, 2011 Revised March 1, 2012 Accepted March 2, 2012 Research Article pH Fractionation and identification of proteins: Comparing column chromatofocusing versus liquid isoelectric focusing techniques In proteomic investigations, a number of different separation techniques can be applied to fractionate whole cell proteomes into more manageable fractions for subsequent analysis. In this work, utilizing HPLC and mass spectrometry for protein identification, two different fractionation methods were compared and contrasted to determine the potential of each method for the simple and reproducible fractionation of a bacterial proteome. Column- based chromatofocusing and liquid-based isoelectric focusing both utilized pH gradients to produce similar results in terms of the numbers of proteins successfully identified (402 and 378 proteins) and the consistency of proteins identified from one experiment to the next (<10% change). However, there was limited overlap in the protein sets with <50% of the proteins identified as common between the sets of proteins identified by the different systems. In addition to the numbers of proteins identified and consistency of those identified, the reduced monetary costs of experimentation and increased assay flexibility produced by using isoelectric focusing was considered in order to adopt a system best suited for comparative proteomic projects. Keywords: Chromatofocusing / Escherichia coli / Isoelectric focusing DOI 10.1002/jssc.201100865 1 Introduction How bacteria, including human pathogens, survive and per- sist within varied environments and cause disease is depen- dent on the complement of proteins the bacteria are able to produce at any given time. To understand bacterial mech- anisms, we must be able to identify and measure the pro- teins produced by the organism under clearly defined con- ditions. Well-defined reverse-phase liquid chromatography- mass spectrometry based techniques commonly known by the acronym MudPIT (Multidimensional protein identifica- tion technology) for the identification of large collections of proteins exist and are readily applicable to bacterial re- search [1]. However, the expected size of bacterial proteomes can be relatively large; a bacteria like Escherichia coli is ex- pected to be capable of expressing greater than 4000 dif- ferent proteins [2]. For this reason, it is in some condi- Correspondence: Dr. Nereus W. Gunther IV, Eastern Regional Research Center, Molecular Characterization of Foodborne Pathogens Research Unit, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA E-mail: jack.gunther@ars.usda.gov Fax: +215-233-6581 Abbreviations: CB, column based; CF, chromatofocusing; LB, liquid based; LC-MS, liquid chromatography mass spectrom- etry; PLGS, ProteinLynx global server; UPLC, ultra pressure liquid chromatographer tions preferable to select a portion of the entire proteome from which to identify and measure the quantity of pro- teins contained within the fraction. There are several dif- ferent well described techniques for sub-dividing a bacte- rial proteome for investigation. Some techniques preferen- tially isolate a sub-cellular class of proteins from a sam- ple, such as membrane proteins or cytoplasmic proteins [3]. Other techniques isolate the entire proteome but then fractionate it into more manageable units with fewer num- bers of total proteins in the subsequent fractions [4–8]. The most common technique used to fractionate a pro- teome is 2-D gel electrophoresis, which utilizes isoelec- tric focusing based on the isoelectric point (pI) of the in- dividual proteins as the first separation step [9–12]. Iso- electric focusing is often conducted using immobilized pH gradients on gel strips [13, 14]. However, the separa- tion of proteins based on pI can also be accomplished using column-based (CB) chromatofocusing (CF) [15] and liquid-based (LB) isoelectric focusing (IEF) [16–27]. An ad- vantage of using CB-CF or LB-IEF is that the proteins are separated and eluted in liquid state, making down- stream techniques such as liquid chromatography-based ∗ N. W. Gunther IV, M. Paul, and A. Nu˜ nez contributed equally to the completion of the presented research. 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