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Isoelectric Point Separations of Peptides and Proteins 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. Two Two proteins proteins with with varying varying isoelectric isoelectric points points will will migrate migrate in inthe the presence presence of of a apH pH gradient gradient and and electric electric field field until until the the net net charge charge of of a proteinprotein isis zero,zero, inin whichwhich migrationmigration willwill cease.cease. CarrierThe history ampholytes of IEF begins are the with most early commonly work carried used chemical out by A.J.P. components Martin [1] used who to made generate several pH gradients.contributions The chemistryin the field of of carrier electrophoresis. ampholytes Martin was originally also contributed generated viasignificantly pentaethylenehexamine to the field of andchromatography addition of acrylic and was acid. awarded A second a Novel generation Prize for approach his efforts. in The carrier work ampholyte of P.G. Righetti synthesis has been was performedparamount by in Vesterberg the ability [5], to in separate which a heterogeneousbiomolecules mixtureelectrophoretically, of amines was particularly reacted with according acrylic acid to andisoelectric a complex point. product To fully resulted understand in the generation these contributions, of thousands one of molecules must review with varyingthe details pI values,of the yetexperiment, very small particularly changes in pI establishing values across the a pH pH range. gradient. Therefore, Furthermore an ideal carrier, classical ampholyte work mixtureregarding is generated—aampholytes was large carried number out of by components Svensson in with the closeearly pI 1960s values [2– resulting4]. in a linear pH gradient. With regardCarrier to gels, ampholytes carrier ampholytes are the most can also commonly be embedded used intochemical acrylamide components gels and used separation to generate carried pH outgradients. in slab/flatbed The chemistry format. of Details carrier regardingampholytes the was specifics originally of carrier generated ampholyte via pentaethylenehexamine synthesis and history haveand beenaddition previously of acrylic reviewed acid. A [6 ,second7]. generation approach in carrier ampholyte synthesis was performedA major by achievement, Vesterberg [5] which, in which was ana heterogeneous extension of the mixture synthesis of amines of carrier was ampholytes, reacted with was acrylic the generationacid and a of complex immobilized product pH resulted gradients in in the 1982 generation [8]. Bjellqvist of thousands et al. utilized of molecules acrylamide with as avarying backbone pI incorporatingvalues, yet very amino small and changes carboxyl in pI groups values via across radical a pH mediated range. reactionsTherefore, allowing an ideal forcarrier branching ampholyte and crosslinkingmixture is generated with carrier—a ampholyteslarge number of differentof components pKa values. with close The resultingpI values product resulting is ain pH a linear gradient pH thatgradient. is immobile With regard in an electric to gels, field carrier and ampholytes acts as a buffer. can Thealso valuesbe embedded of pH range into fromacrylamide 1 to 13 gels and canand beseparation synthesized carried in linear out in and slab/flatbed nonlinear format. forms. TheDetails length regarding of the IEFthe setupspecifics that of is carrier used plays ampholyte a role insynthesis the desired and resolutionhistory have needed. been previously This major reviewed advancement [6,7]. opened doors for various applications of isoelectricA major focusingachievement, for the which separation was an of extension biological ofmolecules, the synthesis especially of carrier peptides ampholytes, and proteins. was the Thegeneration resolving of powerimmobilized of IEF pH (DpI) gradients is determined in 1982 by [8] a. Bjellqvist series of factors et al. utilized in the experimentacrylamide includingas a backbone the diffusionincorporating coefficient, aminoconductivity and carboxyl and groups the electricvia radical current mediated density. reactions Properties allowing of the for gradient branching include and thecrosslinking slope and with the charge carrier curve ampholytes near the of focusing different point. pKa values. These propertiesThe resulting and product relationships is a pH have gradient been reviewedthat is immobile in detail in [6 an,9]. electric field and acts as a buffer. The values of pH range from 1 to 13 and can be syntIEFhesized can be performedin linear and in nonlinear a variety offorms. formats, The includinglength of the preparative, IEF setup analyticalthat is used and plays microscale. a role in Onthethe desired larger resolution end, IEF has needed. proven This to be major beneficial advancement as a preparative opened method doors duefor tovarious its ability applications to separate of largeisoelectric amounts focusing of samples for the providing separation high of resolution biological with molecules, large recovery especially yields. peptides Notably, and this proteins. separation The methodresolving is advantageouspower of IEF in(ΔpI) its abilityis determined to concentrate by a series large quantitiesof factors ofin samplesthe experiment while simultaneously including the removingdiffusion coefficient, common interfering conductivity agents and or the unwanted electric current analytes. density. Additionally, Properties IEF of can the begradient carried include out in capillaries,the slope and microfluidic the charge channelscurve near and the multi-compartment focusing point. These electrolyzers properties and (MCE) relationships as described have below. been Inreviewed general, in IEF detail is an [6,9 extremely]. powerful technique that, when used in any format, allows for the fractionationIEF can ofbe samplesperformed resulting in a variety in reduced of formats, sample including complexity preparative, and more analytical in-depth analysis. and microscale. On the larger end, IEF has proven to be beneficial as a preparative method due to its ability to separate large amounts of samples providing high resolution with large recovery yields. Notably,
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