proteomes Review Isoelectric Point Separations of Peptides and Proteins Melissa R. Pergande and Stephanie M. Cologna * Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-312-996-3161 Academic Editors: Jens R. Coorssen, Alfred L. Yergey and Jacek R. Wisniewski Received: 22 October 2016; Accepted: 8 January 2017; Published: 25 January 2017 Abstract: The separation of ampholytic components according to isoelectric point has played an important role in isolating, reducing complexity and improving peptide and protein detection. This brief review outlines the basics of isoelectric focusing, including a summary of the historical achievements and considerations in experimental design. Derivative methodologies of isoelectric focusing are also discussed including common detection methods used. Applications in a variety of fields using isoelectric point based separations are provided as well as an outlook on the field for future studies. Keywords: isoelectric focusing; proteomics; electrophoresis; two-dimensional gel electrophoresis; isoelectric trapping; capillary isoelectric focusing 1. Introduction The separation of biomolecules, particularly proteins, in the presence of an electric field (e.g., electrophoresis) has given rise to an array of methodologies to reduce the complexity of samples to probe the physiochemical properties of such biomolecules. Proteins and peptides represent possibly the most highly studied class of molecules that are interrogated by electrophoretic methods. These methods include: agarose and polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis (2DE), capillary electrophoresis, isotachophoresis and others. One such electrophoretic technique is isoelectric focusing (IEF) which provides separation of ampholytic components, molecules that act as weak acids and bases, according to their isoelectric points. In IEF, ampholytes travel according to their charge under the influence of an electric field, in the presence of a pH gradient, until the net charge of the molecule is zero (e.g., isoelectric point, pI). When considering peptides and proteins, the separation is deemed according to the composition of amino acids and exposed charged residues, which behave as weak acids and bases (Figure1). The migration of the ampholytic species will follow basic principles of electrophoresis; however, the mobility will change in the presence of the pH gradient by slowing down migration at values close to the pI value. Even the simplest ampholytes (e.g., amino acids) can create a pH gradient and act as an isoelectric buffer. The history of IEF begins with early work carried out by A.J.P. Martin [1] who made several contributions in the field of electrophoresis. Martin also contributed significantly to the field of chromatography and was awarded a Novel Prize for his efforts. The work of P.G. Righetti has been paramount in the ability to separate biomolecules electrophoretically, particularly according to isoelectric point. To fully understand these contributions, one must review the details of the experiment, particularly establishing the pH gradient. Furthermore, classical work regarding ampholytes was carried out by Svensson in the early 1960s [2–4]. Proteomes 2017, 5, 4; doi:10.3390/proteomes5010004 www.mdpi.com/journal/proteomes Proteomes 2017, 5, 4 2 of 14 Proteomes 2017, 5, 4 2 of 13 FigureFigure 1.1. PrinciplePrinciple of isoelectric focusing. 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