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Review Article *Corresponding author Brian K. Kay, Department of Biological Sciences, University of Illinois at Chicago, Ashland, Chicago, IL, Reagents for Detecting USA, Tel: 13123550668; Email: Submitted: 11 September 2017 Phosphosites within Accepted: 16 October 2017 Published: 18 October 2017 1 2 2 Leon A. Venegas , Qi Zhao , Michael P. Weiner , and Brian K. Copyright Kay1* © 2017 Kay et al. 1 Department of Biological Sciences, University of Illinois at Chicago, USA OPEN ACCESS 2Abcam, USA Keywords Abstract • Affinity reagents • Phosphorylation phosphorylation is an important post-translational modification [PTM] • Phos-tag that cells employ to coordinate diverse biological processes. In the past, • pIMAGO have been used as tools for studying phosphosites within proteins, but due to intrinsic limitations with their production and validation, scientists are now turning to other options. Two types of new reagents have recently emerged as attractive alternatives for monitoring phosphosites: polymers [i.e., pIMAGO, Phos-tag] and recombinant affinity reagents [i.e., DARPINs, FHA domain, FN3, scFv]. Both types of reagents can be used in ELISA, western blotting, and cell staining experiments to detect phosphorylated proteins. This mini-review focuses on the development and utility of each of their agents for detecting phosphosites and examines their strengths and weaknesses.

ABBREVIATIONS CDR: Complementarity-Determining Region; DARPins: critical, as altered expression or activity of protein kinases [11], Designed Ankyrin Repeat Proteins; ELISA: Linked phosphatases [12] or mutation of the site of phosphorylation Immunosorbent ; ERK: Extracellular Signal-Regulated techniques[13] are linked and totools a variety for quantitative of diseases and [14]. qualitative With over studies 100,000 of Kinase; FHA: Forkhead-associated; FN3: Fibronectin Type III; reported phosphosites [15], there is a substantial need for FRET: Fluorescence Resonance Energy Transfer; H: Heavy phosphorylationThe current methodsevents. for studying protein phosphorylation Polypeptide Gene Enhancer In B-Cells Inhibitor, Alpha; L: Light Chain of Immunoglobulin; MAB: Iκbα: NuclearMonoclonal Factor ; of Kappa MALDI- Light 32 TOF MS: Matrix-Assisted Laser Desorption/Ionization Time- are mass spectrometry [16], P-labeling [17], enzyme linked of-Flight Mass Spectrometry; MEK: Mitogen-Activated Protein immunosorbent assays (ELISA) [18], ATP consumption [19,20], Kinase; pAb: Polyclonal Antibody; PTM: Post-Translational fluorescence based assays [21], fluorescence polarization [22, and23], fluorescencelimitations for resonance providing energy insight transfer into the (FRET) regulation [24], and of SPR: Surface Plasmon Resonance; tRNA: transfer Ribonucleic fluorescence quenching [25]. Each method has its advantages AcidModification; scFv: Single-Chain Fragments of Variable Regions; biological reagents for studying protein phosphorylation, in lieu phosphorylation. This review will focus on use of chemical and INTRODUCTION the functional utility of both traditional and non-traditional Protein phosphorylation is an important post-translational of mass spectrometry. It will cover affinity reagent generation, STUDYING PROTEIN PHOSPHORYLATION WITH With 30% of a cellular proteins estimated to be phosphorylated reagents, and progress in reagent production. modification (PTM) implicated in regulating cellular activities [1]. PHOSPHOSPECIFIC ANTIBODIES Antibodies are commonly used tools for probing proteins in at any given time [2], this PTM is utilized in diverse ways to coordinate protein function [3]. Examples include providing docking sites to allow for protein-protein interactions [4], complex mixtures and cells. Antibody production and validation regulation of activity and subcellular localization [5], marking is a long (i.e., 3-12 months) and rigorous process (Figure 1). To proteins for degradation [6], and activating or inactivating produce an antibody, a purified or recombinant target protein is proteinsMost in eukaryoticcell signaling proteinspathways [2]. contain more than one injected into an animal host, which triggers an immune response and subsequent production of antibodies. Polyclonal antibodies immortalized(pAbs) are purified B cell of from the imm the serum of the immunized animal, phosphorylatable site [phosphosite], which primarily occurs on whereasalso be generated monoclonal to s ynthetic antibodies peptides (mAbs) corresp are secretedonding to frompeptide an serine (89%), threonine (10%), and tyrosine (<1%) residues unized animal. pAbs and mAbs can [7-10]. Elucidating the functional role of phosphosites is

Cite this article: Venegas LA, Zhao Q, Weiner MP, Kay BK (2017) Reagents for Detecting Phosphosites within Proteins. J Bioinform, Genomics, Proteomics 2(3): 1022. Kay et al. (2017) Email:

Central Bringing Excellence in Open Access segments of a protein that incorporate a phosphor NEW REAGENTS FOR DETECTING PROTEIN For example, to produce an antibody against phosphorylated PHOSPHORYLATION ylated residue. pT Investigators have turned their attention to developing human Akt1, which carries a phosphothreonine at position 308 Serum(i.e., pT3080), from the a 15-mer immunized peptide animal (KDGATMK can be chromatographedFCGTPE) would polymers and recombinant affinity reagents for monitoring overbe synthesized a column and containing used as the the immobilized immunogen phosphopeptide,(with adjuvant). demonstrated great utility in detecting phosphorylated targets phosphorylation. These alternative technologies have systems bind the phosphoryl moiety and can be used to quantify and antibodies that recognize the peptide are then affinity with a high level of accuracy. Both the Phos-tag and pIMAGO purified. If desired, a subsequent subtraction step can be used the level of phosphorylation within a protein or cell lysate. antibodiesto eliminate can antibodies be chromatographed from serum over that across-react column containing with the Recombinant affinity reagents offer the same advantages of thenon-phosphorylated non-phosphorylated form form of the of the peptide; peptide the attached affinity to purified resin, antibodies, but their affinities and specificities can be readily manipulatedDETECTION through OF molecular PHOSPHOEPITOPES biology techniques. USING THE PHOS-TAG SYSTEM yielding antibodies (in the flow-through) that recognize the andpeptide after sequence several rounds only when of injections, phosphorylated. splenocytes To generate from the mAbs, host an animal host is injected with the phosphopeptide of interest, The Phos-tag (Figure 2) was developed to identify are isolated and B cells fused with immortalized myeloma cells to phosphorylation and monitor kinase activity without the need produce hybridomas. Hybridoma clones are plated into microtiter Phos-tagenhancesof phosphospecific detection antibodies of or phosphopeptidesinradiolabeling [43, 44]. matrix- The plate wells, and cells that secrete the desired phosphospecific reagent can be used in a variety of experimental formats. The antibody are then cloned. By either method, it is possible to generate antibodies that bind the peptide sequence only when assisted laser desorption/ionization time-of-flight mass phosphorylatedWhile pAbs (i.e., and the mAbs phosphate can be veryis part effective of the ). in monitoring spectrometry (MALDI-TOF MS). Phosphorylation of proteins can be also monitored with this reagentin surface plasmon resonance (SPR) [45], SDS-PAGE [46-51], western blots [43], and FRET pAbsphosphorylation and mAbs are events raised by againstwestern a blotphosphopeptide; [26-30] and cell there staining is no [52]experiments. Finally, the Phos-tag can be used in affinity [31], there are several limitations to these reagents. First, when onechromatography limitation to [53,note 54] about to purify the Phos-tag phosphopeptides is the inability in bulk of from this a trypsin-digested cell lysate for biochemical analysis. However, assurance that such antibodies will recognize the native, folded qualityform of of thepAbs phosphoprotein. and mAbs is not Consequently,completely reproducible they may from not lot be reagent to differentiate between distinct phosphosites within a useful for pull-down and cell staining experiments. Second, the protein.DETECTION WITH NOVEL POLYMERS to lot [32]: immune responses vary between animals, which leads nanopolymer conjugated to titanium ions that can bind phosphate to batch-to-batch differences [33], and hybridomas may stop The pIMAGO system (Figure 2) is a water-soluble secreting mAbs due to genetic drift [34,35] or clonal instability [36]. Third, there are many examples of cross-reactive antibodies [55-58]. pIMAGO reagents can be tagged with an infrared in[37-42], chemical requiring and protein immunization engineering of have additional provided animals alternative until sizefluorescence of the polymer (IR) dye is oneor biotin of its mainfor detection strengths; of itphosphoproteins is small enough approachesthe desired specificity for overcoming is attained. the Fortunately, limitations of advancements generating in dot blots [59], western blots [60], or ELISAs [57]. The small production times, eliminate the need for animals, ensure Thisto be is used advantageous simultaneously because in a steric western hindrance or dot can blot decrease with an antibodies to phosphoepitopes. These methods provide shorter antibody that recognizes a different epitope in the same protein. renewability and offer improved specificity. simultaneous binding by two different immunoglobulins, which

2-4 months

Figure 1

A pipeline to produce phosphospecific reagents. There are two approaches for generating phosphospecific reagents. The first is that an animal is injected with a phosphopeptide to produce antibodies (i.e., mAb, pAb). The reagents then undergo a series of quality control (Q.C.) checks before they are tested in assays. Failure to pass Q.C. checkpoints results in the process having to be repeated. Alternatively, in display technologies, a phosphopeptide is used as a target in affinity selection, binding variants are isolated, and affinity matured to produce a “super binder,” which can be used in assays. These strategies take between one and 12 months. J Bioinform, Genomics, Proteomics 2(3): 1022 (2017) 2/7 Kay et al. (2017) Email:

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Figure 2

Phosphoprotein detection by antibody alternatives. A. Phos-tag (left) is a binuclear metal (II) complex of 1,3-bis [bis (pyridine-2-ylmethyl)amino]propan-2-olate that interacts with the phosphorylmoiety. The Phos-tag has a vacancy on two metal ions that is suitable for accessing the monoester dianion as a bridging ligand. pIMAGO (right) is a dendrimer conjugated to titanium ions (yellow arrows) that bind the phosphoryl moiety. Each molecule can be tagged with biotin (blue circle). B. Detection of the phosphoprotein can be achieved by following the binding of biotinylated Phos-tag or pIMAGO to a protein species, followed by detection with horseradish peroxidase (HRP) conjugated to avidin. Not to scale.

Figure 3

Structural model of an scFv in complex with a phosphopeptide. Left: Alanine scanning of CDR H2 and % peptide-binding activity as compared to the wild type antibody. Right: The electrostatic contour of the scFv surface displays a deep negative charged groove connecting to H2; blue, grey, and red surfaces represent, positive, neutral, and negative residues, respectively. The positively charged half of the phosphopeptide (MARRPRHSIYpSSDEDDEDFE, where pS is phosphoserine) is shown in stick form bound to an scFv; blue, orange, red, and yellow represent nitrogen, phosphorus, oxygen, and carbon, respectively. The peptide was manually modelled in the groove and refined using Rosetta Flex Pep Dock (http://flexpepdock.furmanlab.cs.huji.ac.il/).

in vitro are 150 kDa in size. Simultaneous binding of the pIMAGO advantages over animal produced antibodies. Since recombinant reagent and an antibody allows one to quantify the amount of affinity reagents are generated in or through phosphorylated protein amino relative acids, the to the reagent total amount cannot of be the used target. to methods [61], there is no need for animals. Plus, their DNA However, as the pIMAGO reagent binds equally well to different sequence is known, allowing them to be shared electronically includebetween theirresearch ability groups to beand tagged easily ordered through as molecular synthetic DNA biology for identifyDETECTION the actual WITHphosphosite. RECOMBINANT AFFINITY insertion into a suitable expression vector [62]. Other advantages REAGENTS techniques with different epitope sequences or fusion partners, without interfering with their binding properties [63], and their Recombinant affinity reagents offer a number of distinct ability to yield tight, specific binders through J Bioinform, Genomics, Proteomics 2(3): 1022 (2017) 3/7 Kay et al. (2017) Email:

Central Bringing Excellence in Open Access in vitro subtraction of permits approachesprotein conformations, [64]. Finally, individual members of a protein family, well as the proven design of phosphate binding sites altogether generation of recombinant affinity reagents that are specific for make a highly focused, knowledge-based phospho-status binding 8 library of scFvs possible. Using a combination of functional ordisplay PTMs technologies [62,65]. Large such (i.e.,as phage-, >10 ) yeast-, libraries ribosome-, of recombinant mRNA-, mutagenesis and structural modeling as well as a comparison affinity reagents can be screened against targets of interest using with existing structures of phospho-peptide Ab complexes, we ts and non-antibody scaffold have identified a shared peptide binding groove holstered by and bacterial-display [66]. Two common types of recombinant several restricted regions of CDR L3 and H3, which can serve as a affinity reagents, antibody fragmen suitableScaffold candidate proteins for a arefocused good phosphopeptide alternatives to library recombinant design. proteins,Antibody are describedfragments, below. such as human single-chain Fragments antibodies as they can imitate antibody binding to their target. of variable regions (scFv) can be displayed on the minor coat Scaffold proteins are typically small, well expressed in bacterial protein III (P3) of the and used in phage and other hosts, lack disulfide bonds, thermal stable, and display affinity selection experiments to isolate phosphospecific highly soluble. Three types of scaffolds have been successfully binders. For example, a 15-mer peptide is chemically synthesized engineered to recognize phosphoepitopes: the fibronectin type III with a phosphoresidue at its center, and then used in affinity (FN3) monobodies, designed ankyrin repeat proteins (DARPins), selection experiments. Typically, three rounds of affinity andThe the Forkhead FN3 scaffold Associated has been (FHA) used domain. to isolate variants that selection are sufficient to recover binding clones from a display can bind a phosphopeptide from nuclear factor of kappa light oflibrary, binders and are their often binding converted properties to immunoglobulins [i.e., affinity, specificity] for stability can be improved through mutagenesis, if necessary. The final sets polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα). A library of the FN3 monobodies was mRNA displayed and affinity and avidity. Phosphospecific-scFvs have been isolated by phage phosphorylatedselected against peptide, a phosphorylated had a K peptide target from IκBα. display against a panel phosphopeptide targets [67], including a d The resulting FN3 domain was shown to be specific for the phosphohistidine-containing Phosphate binding sites target in scFvs [68]. can be designed as a in vitro of 18 nM, and was shown to recognize endogenous IκBα by and detected the oflocalized an Ab module. to bind phosphorylatedResearch published amino by both acids Koerber is dependent [69] and on products of IκB kinase [73]. Shih [67], and work by the Weiner lab, suggest that the ability A library of DARPins was ribosome displayed and affinity selected against fully folded ERK1/2 protein that were either the complementarity-determining region (CDR)2 of the heavy phosphorylated (ERK1/2-pTpY) or non-phosphorylated. The and/or light chains. CDR2 makes direct contacts with the chains playing a role in the directionality of the phosphopeptide selected reagents could distinguish between the states of the phosphorylated-, with CDR2 of the light or heavy andkinase bioluminescence through ELISA. resonance To test the energy functional transfer utility experiments of the ERK panel of DARPins, the reagents were used in [74] bindingas it fits incan the be scFv’s incorporated groove. Thus, into the instead phage of display random library mutagenesis design of all six CDR residues, prior knowledge of phospho-amino acid to showcase the level of specificity for the reagents . The ERK1/2-pTpY specific DARPins could be converted into to produce a library with a higher frequency of modification- for binding phosphorylated targets using three constrained biosensors and were able to detect the subcellular site of ERK1/2 specific relevant binders. Such a phospho-focused library phosphorylationAlternatively, in one living can cellsuse a [75]. scaffold that naturally recognizes 3 phospho-interacting amino acid positions will be effectively naturally recognizes a phosphothreonine site on its target 8,000both the (i.e., phosphorylated 20 ) fold more amino efficient, acid andvaluable the surrounding and practical context over a phosphopeptide residue. For example, the FHA1 domain randomized libraries. Phosphospecific Abs generally recognizes protein, pRad9. Through directed evolution, a phage library of sequence. The successful phosphorylated peptide Abs usually phosphorylatedthermal stable FHA1 phosphothreonine-containing variants was generated [76] peptide and affinitytargets contextrecognize sequence 4-5 extra might residues be used upstream to offset orthe downstream contribution ofof the selection with a variety of mono-phosphorylated or dual- phosphorylated aminoresidue acid [67,69], and consequently suggesting excess enable binding phospho- to independent peptide binding in a subsequent variant of a [76-79] yield recombinant affinity reagents to 14 out of 17 targets, reflecting a 82% success rate. All isolated FHA domains were shown to be both phosphothreonine (pThr)-dependent and recombinant scFv. specific, as compared to commercial antibodies raised against the sameLike phosphopeptide all technologies, [79]. the uses of scaffold proteins to Based on our alanine scanning results (Figure 3) and our monitor protein phosphorylation have their advantages and and Koerber’s findings that H2 is the phosphate-binding center [69], we modeled the Ab structure of our antibody AXM1293 and its complex with its cognate phosphorylated peptide, in disadvantages. As a recombinant protein, the DNA sequence collaboration with Dr. Jeffrey Gray [70-72]. Through comparison mutationsof the scaffold of the is scaffold known, canand be mutations created canthat be destroy introduced binding; to with published structures, we noticed that L2 and H2 can be enhance binding strength or specificity. Additionally, point utilized to target phosphopeptides with C-terminal or N-terminal Finally, the recombinant scaffolds can be expressed inside cells context sequences, respectively. such variants make excellent negative controls in experiments. The uniform structure, the likely defined context length, as J Bioinform, Genomics, Proteomics 2(3): 1022 (2017) 4/7 Kay et al. (2017) Email:

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o guarantee that an isolated Signaling Components, Nuclear Roles and Mechanisms of Nuclear where they can interfere the phosphorylated form of the target. 5. Plotnikov A, Zehorai E, Procaccia S, Seger R. The Mapk Cascades: One major drawback is that there is n Translocation. Biochim Biophys Acta. 2011; 1813: 1619-1633. binder will be able to the phosphorylated target in its native, C-Myc Protein Stability through Opposing Phosphorylation Events folded state. However, one can screen many clones and identify 6. Escamilla-Powers JR, Sears RC. A Conserved Pathway That Controls thoseCONCLUDING that recognize REMARKS the target in the native state. Occurs in Yeast. J Biol Chem. 2007; 282: 5432-5442. 7. Lin J, Xie Z, Zhu H, Qian J. Understanding Protein Phosphorylation on a This review presents recent progress in developing reagents Systems Level. Brief Funct Genomics. 2010; 9: 32-42. for monitoring protein phosphorylation. Phosphospecific 8. Ubersax JA, Ferrell JE. Mechanisms of Specificity in Protein antibodies, pIMAGO, Phos-tag, and recombinant affinity reagents Phosphorylation. Nat Rev Mol Cell Biol. 2007; 8: 530-541. have shown their merit for advancing our understanding of signaling events. With these advancements, it is possible to follow 9. Endicott JA, Noble ME, Johnson LN. The Structural Basis for Control of Eukaryotic Protein Kinases. Annu Rev Biochem. 2012; 81: 587-613. and quantify phosphorylation of proteins within cells and tissues. 10. Mann M, Ong SE, Gronborg M, Steen H, Jensen ON, Pandey A. Analysis In the future, it will be important to use folded phosphorylated of Protein Phosphorylation Using Mass Spectrometry: Deciphering proteins instead of phosphopeptides as targets. As previously the Phosphoproteome. Trends Biotechnol. 2002; 20:‘at Larg261-268.e duementioned, to the inabilitythese two to classesproduce of native reagents proteins are selected phosphorylated by using phosphopeptides as targets. 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Cite this article Venegas LA, Zhao Q, Weiner MP, Kay BK (2017) Reagents for Detecting Phosphosites within Proteins. J Bioinform, Genomics, Proteomics 2(3): 1022.

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