Chemical Approaches to Investigate Labile Peptide and Protein

Article pubs.acs.org/accounts

Chemical Approaches to Investigate Labile Peptide and Protein Phosphorylation Published as part of the Accounts of Chemical Research special issue “Chemical Biology of Peptides”. Anett Hauser,†,‡ Martin Penkert,†,‡ and Christian P. R. Hackenberger*,†,‡ †

Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Robert-Roessle-Straße 10, 13125 Berlin, Germany Institute of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Straße 2, 12489 Berlin, Germany

‡

CONSPECTUS: Protein phosphorylation is by far the most abundant and most studied post-translational modification (PTM). For a long time, phosphate monoesters of serine (pSer), threonine (pThr), and tyrosine (pTyr) have been considered as the only relevant forms of phosphorylation in organisms. Recently, several research groups have dedicated their efforts to the investigation of other, less characterized phosphoamino acids as naturally occurring PTMs. Such apparent peculiar phosphorylations include the phosphoramidates of histidine (pHis), arginine (pArg), and lysine (pLys), the phosphorothioate of cysteine (pCys), and the anhydrides of pyrophosphorylated serine (ppSer) and threonine (ppThr). Almost all of these phosphorylated amino acids show higher lability under physiological conditions than those of phosphate monoesters. Furthermore, they are prone to hydrolysis under acidic and sometimes basic conditions as well as at elevated temperatures, which renders their synthetic accessibility and proteomic analysis particularly challenging. In this Account, we illustrate recent chemical approaches to probe the occurrence and function of these labile phosphorylation events. Within these endeavors, the synthesis of site-selectively phosphorylated peptides, in particular in combination with chemoselective phosphorylation strategies, was crucial. With these well-defined standards in hand, the appropriate proteomic mass spectrometry-based analysis protocols for the characterization of labile phosphosites in biological samples could be developed. Another successful approach in this research field includes the design and synthesis of stable analogues of these labile PTMs, which were used for the generation of pHis- and pArg-specific antibodies for the detection and enrichment of endogenous phosphorylated samples. Finally, other selective enrichment techniques are described, which rely for instance on the unique chemical environment of a pyrophosphate or the selective interaction between a phosphoamino acid and its phosphatase. It is worth noting that many of those studies are still in their early stages, which is also reflected in the small number of identified phosphosites compared to that of phosphate monoesters. Thus, many challenges need to be mastered to fully understand the biological role of these poorly characterized and rather uncommon phosphorylations. Taken together, this overview exemplifies recent efforts in a flourishing field of functional proteomic analysis and furthermore manifests the power of modern peptide synthesis to address unmet questions in the life sciences.

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INTRODUCTION Proteins are involved in virtually every cellular mechanism, including biosynthesis, DNA transcription and translation, as well as molecular trafficking and signaling, thereby serving as either substrate or enzyme catalyst. With an estimated number of 3−4 million proteins per cell in Escherichia coli and approximately 100−150 billion in mammalian cells,1 the huge diversity in form and function can only be achieved by posttranslational modification (PTM) of proteins. Protein phos© 2017 American Chemical Society

phorylation of nucleophilic residues is by far the most abundant PTM, indeed to an extent that every third protein is believed to be phosphorylated at least once in its lifetime.2 Over the years, researchers have focused primarily on studying the phosphorylation of the hydroxyl groups of serine (Ser), threonine (Thr), and tyrosine (Tyr), which was accompanied by revolutionary Received: April 5, 2017 Published: July 19, 2017 1883

DOI: 10.1021/acs.accounts.7b00170 Acc. Chem. Res. 2017, 50, 1883−1893

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Accounts of Chemical Research

Figure 1. Fascinating but poorly characterized amino acid phosphorylations with reports indicated for their in vitro (blue) or in vivo (green) detection. Phosphoramidates: pHis (two isomers), pArg, pLys, and polyP Lys; phosphorothioate: pCys; anhydrides: pAsp, pGlu, ppSer, and ppThr.

advances in phosphoproteomic analysis such as modern mass spectrometry methods,3 the availability of phosphorylationspecific antibodies,4 as well as the development of cell-based tools5 and semisynthetic protein synthesis6 to study the function of phosphorylated proteins. In addition, other less common post-translational phosphorylations have recently been discovered. These occur on other nucleophilic side chains, such as the nitrogen-containing functionalities of histidine (His), arginine (Arg), and lysine (Lys), the thiol group of cysteine (Cys), and the carboxylate anions of aspartic acid (Asp) and glutamic acid (Glu), resulting in phosphoramidates, phosphorothioate, or mixed anhydrides.7−10 Already phosphorylated Ser and Thr residues can also be further phosphorylated to pyrophosphates (Figure 1).11 Phosphorylated nucleophilic amino acids other than Ser, Thr, and Tyr have limited chemical and thermal stability. Most are highly labile in acidic media as well as at elevated temperatures but often rather stable at basic pH. This stability profile results in an underdeveloped level of knowledge about their biological significance, especially because routine methods for isolation, detection, and characterization of pSer-, pThr-, or pTyrcontaining proteins are often too harsh. Hereby, the main drawback is that conventional protocols often contain at least one step of applying acidic conditions under which the phosphate group would be cleaved off and hence not detected in further analysis. Several research groups have risen to the challenge to elucidate the poorly characterized phosphorylations by developing convenient chemical and analytical methods selective for each of those defiant phosphoamino acids. In the course of these analyses, taking care and advantage of specific chemical properties is one of the main characteristics of this work. We present here a number of recent approaches by our group and others to elucidate the biological role of

peculiarly phosphorylated amino acids, in particular pHis, pArg, pLys, pCys, ppSer, and ppThr. Besides these six types of phosphorylation, polyphosphorylation of Lys (polyP Lys) and anhydride formation of Glu and Asp are also known, but for those, no recent synthetic or proteomic approaches have been reported. Polyphospate (polyP) Lys was described by the Saiardi group in 2015.12 They could demonstrate via mutagenic studies that, in yeast cells, chains of 32P-inorganic polyP can be covalently bound to the two interacting enzymes Nsr1 and Top1. In both cases, polyP Lys was observed in polyacidic lysine-rich clusters that were initially assumed to be pyrophosphorylated on Ser. Because inositol pyrophosphates regulate ATP as well as polyP levels in cells, they are also responsible for serine pyrophosphorylation and indirectly for polyP Lys formation. It would be interesting to know if these two PTMs interact. To date, no chemical synthesis of polyP Lys, stable analogues, or proteomic approaches for this modification are known. Acyl phosphates in pAsp or pGlu are known to occur as free intermediates during amino acid biosynthesis and in the active sites of enzymes. These PTMs are commonly studied, e.g., using colorimetric detection with FeIII, chromatographic comparison, or selective reduction with sodium borohydride and subsequent thin layer chromatography; however, for pAsp or pGlu, no reports on novel synthetic approaches have been published recently.13

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ROADMAP TO ANALYTICAL PROTOCOLS Disparate approaches are possible to elucidate highly attractive but incomprehensibly studied, labile, post-translational phosphorylations. For example, one could start hunting for those PTMs by adjusting conventional proteomic bottom-up approaches to less acidic conditions and applying typical mass spectrometry (MS) fragmentation techniques on analytical 1884


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that take advantage of distinctive reactivities of amino acid residues site specifically, phosphorylated peptides could be obtained in several cases.16−18 Subsequently, these model peptides delivered information regarding their stability in different media, their chromatographic behavior, and their fragmentation pattern under various tandem MS techniques. In this, the main focus is devoted to the conclusive analysis of the PTM of interest, but apart from that, methods to simultaneously evaluate several different modifications are also desired. For selective enrichment, either metal oxides or complexes19−21 of antibodies are regarded as methods of choice. Considering the low intrinsic half-life of P−N, P−S, or P−O−P bonds even under physiological conditions, stable phosphoamino acid mimics are needed to raise specific antibodies as demonstrated previously for pHis-22−26 and pArg-specific27,28 antibodies. With a reliable analytical tool-kit in hand, unknown natural samples of interest will be examined and validated. By conducting such a detailed process for the development of suitable analytic methods, the robustness and reproducibility is assured.

samples. The results will most likely be rather disappointing because conventional MS/MS measurements using collisioninduced fragmentation (CID) may destroy the labile chemical bonds present in phosphoramidates, phosphorothioate, and anhydrides leading to the loss of PTM signal.14 Therefore, further improvements for soft, robust, and sensitive isolation, enrichment, as well as characterization techniques tailored for each PTM are needed. In chemical biology, model systems are designed to establish novel methods for such unmet challenges.15 A typical workflow is shown in Figure 2. The

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SYNTHESIS OF UNCOMMON PHOSPHORYLATED PEPTIDES AND THEIR ANALOGUES For suitable biochemical and proteomic techniques to be developed for the identification of labile PTMs, the availability of site-specific naturally phosphorylated peptides is ideal. By taking advantage of the unique reactivity of an orthogonal functional group in combination with a suitable chemoselective reaction, it is possible to selectively install the desired phosphorylated amino acid within a peptide sequence. The key step in this process is that the chemoselective reaction is performed after solid-phase peptide synthesis (SPPS) with unprotected peptide material, thereby avoiding the exposure of harsh peptide deprotection or cleavage conditions after the installation of the labile phosphorylated amino acid (Figure 3A). Another often pursued strategy in this research area is the generation of antibodies specific for the peculiar phosphorylated amino acid. Because of the intrinsic instability of the phosphoramidate, phosphorothioate, or pyrophosphate, stable analogues are employed in the synthesis of haptens, which are used in the generation of antibodies (Figure 3B).

Figure 2. Workflow to develop and evaluate appropriate analysis protocols for challenging PTMs.

process starts with the design of model systems serving as templates or mimics of natural systems that shall eventually be examined. These model systems need to be defined very precisely so that reliable conclusions can be drawn. When investigating PTMs, highly characterized peptides with specific phosphorylation patterns are required to ensure a reproducible process throughout the analytical method development and validation. Hence, understanding the chemical behavior of the phosphorylated amino acid of interest is a crucial part of the model system. By applying chemoselective synthesis strategies

Figure 3. (A) Chemoselective reactions for the synthesis of peptides with labile phosphorylated residues and (B) use of stable analogues of phosphorylated residues for the generation of specific antibodies. 1885


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Accounts of Chemical Research N-Phosphorylation: pHis, pArg, and pLys

First reports on the synthesis and characterization of phosphorylated N-containing amino acids were already published in the 1960s and 70s (τ-pHis, π-pHis, pArg, pLys; see Figure 1).29−31 Compared to phosphate monoesters (ΔG° = −6.5 to −9.5 kcal/mol),32,33 hydrolysis of phosphoramidates (ΔG° = −10.3 to −14 kcal/mol)34−36 is thermodynamically more likely to occurr. Although the ε-nitrogen in pLys is most likely protonated at physiological pH (pKa of N-butylphosphoramidate is 9.9),37 thereby weakening the P−N bond, the phosphoramidate nitrogens of pHis and pArg are not basic as a result of participation of the lone pair in aromaticity7 and partial delocalization of charge in a formal six-membered ring38 (Figure 4). During the last few years, substantial progress could

Figure 5. (A) Acid-stable analogues of pHis designed for antibody generation24,26,47 and (B) CuI- or RuII-catalyzed cycloaddition to obtain isomeric pHis analogues.22

nopyrazolylethylamine (pPye)26 or 4-phosphopyrazol-2-yl alanine (pPza)47 resulting in highly selective polyclonal Abs; however, no further biological applications were reported for Abs derived from the mentioned analogues. The Hunter group was the first to generate monoclonal antibodies (mAbs) selective against π-pHis or τ-pHis by applying 1-pTza and 3pTza in randomized peptide synthesis.25 With these mAbs in hand, they were further able to study histidine phosphorylation in bacterial40 as well as mammalian cells48,49 and are currently working on developing selective enrichment of pHis peptides for proteomic studies.40

Figure 4. Structural situation in pHis,7 pArg,38 and pLys.37

be accomplished regarding the synthesis of native phosphorylated His, Arg, and Lys monomers and peptides as well as stable analogues, which is outlined below.39

Synthesis of pArg Peptides and Analogues

Synthesis of pHis Peptides and Analogues

After the isolation and characterization of the protein arginine kinase McsB in 1994, thereby validating the occurrence of pArg in prokaryotes for the first time,50 it took more than a decade and the worthwhile effort of the Clausen group to further reveal the mode of action of McsB in more detail.51 Another four years later, the initially annotated pTyr phosphatase YwlE was identified as a pArg phosphatase, thereby being the counterpart for McsB in pArg regulation in bacteria as well as in Drosophila melanogaster.52,53 The latest report indicates that pArg functions as a marker for damaged prokaryotic proteins to induce proteolysis.54 Besides that, the role of McsB in eukaryotes has not yet been completely elucidated.55 Besides phosphorylation with phosphoryl chloride as mentioned above,42 access to pArg as a free amino acid or in peptides can be enabled enzymatically using McsB and ATP.55 For the site-specific installation of pArg in peptides to be achieved, the synthesis of an Fmoc-protected pArg SPPS building block (Fmoc-Arg(PO(OTc)2)-OH) equipped with an acid- and base-resistant 2,2,2-trichloroethyl protecting group was developed by the Seebeck group (Figure 6A).56 Upon hydrogenation at basic pH, the pArg peptides were obtained. In addition to synthesis approaches for natural pArg derivatives, stable analogues were also designed to generate antibodies against pArg. The first example was published by Thompson using (2-((6-aminohexyl)amino)-2-iminoethyl)phosphonic acid (6-pAIE) and 2-((6-aminohexyl)amino)-2iminoethane-1-sulfonic acid (6-sAIE) as stable pArg analogues (Figure 6B).27 Both constructs were applied in the generation of polyclonal antibodies in rabbit, but only the clones derived from the 6-pAIE hapten showed activity against pArg. Furthermore, almost no cross-reactivity with other phosphoamino acids was observed.27 Although 6-sAIE did not prove to be a good pArg mimetic in antibody generation, its sulfonate motif could be successfully applied in YwlE inhibition studies, thus resulting in encouraging IC50 values, whereas derivatives with a phosphonate moiety as in 6-pAIE showed less activity.57 Shortly afterward, 2-((2-ammonioethyl)amino)-2-iminoethyl

Among the often overlooked phosphorylations, pHis is the most studied PTM to date. It is known that this phosphorylation is crucial for prokaryotic signal transduction and as an intermediate for several metabolic enzymes. Furthermore, its importance in mammalian cells has recently increasingly been elucidated.40 Phosphohistidine can be synthesized by reaction of amino acids or unprotected peptides with phosphoryl chloride at pH greater than 9.5.41 Under more basic conditions, lysine31 or in combination with CuII salts, arginine42 residues can be phosphorylated as well. Alternatively, phosphoramidate salts have been reported in residue-selective phosphorylation reactions. For instance, potassium phosphoramidate, which can be obtained by replacement of ammonia from ammonium phosphoramidate, reacts selectively with histidine residues with approximately 15% conversion, whereby π-pHis, τ-pHis (Figure 1), or even 1,3-di-pHis is the favored product depending on the reaction time.41,43 Nevertheless, side reactions with lysine can be expected when the ammonia exchange is not complete.44 It is important to note that these methods do not give access to site-specifically phosphorylated His peptides because specific histidine residues in a given sequence cannot be distinguished. Recently, researchers have devoted more attention to the synthesis of acid-stable pHis analogues for the generation of pan-specific pHis antibodies. All analogues reported so far contain a nonhydrolyzable P−C bond instead of a P−N bond (Figure 5A). With the development of stable triazolylalanine (pTza) derivatives by CuI- or RuII-catalyzed cycloaddition, stable analogues for τ-pHis or π-pHis, respectively, could be obtained22,45,46 (Figure 5B) and applied for the production of the first polyclonal, sequence-dependent anti-3-pHis antibody (Ab) by Muir and co-workers.22 Subsequently, higher affinity for pHis but also considerable cross-reactivity to pTyr of the produced Abs was obtained by employing phosphoryltriazolylethylamine (pTze) as immunogen.24 This issue could be overcome with the second-generation-type analogues phospho1886


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Figure 6. (A) Site-specific synthetic route to pArg peptides56 and (B) acid-stable analogues of pArg for antibody generation.28,57

Figure 7. Chemoselective synthesis of site-specifically phosphorylated Lys peptides with an in-solution approach (A)16 or on a solid support (B).61

As mentioned above, lysine residues can be unselectively phosphorylated with PV reagents41 with no regioselectivity. With the development of the chemoselective Staudingerphosphite reaction60 and its application on azido-lysine peptides, the Hackenberger group recently established a sitespecific synthesis route for pLys peptides in the presence of other lysine residues, which delivered highly valuable peptide material to prime appropriate MS/MS techniques as described later.16,61 By taking advantage of different substituted phosphites, they promoted either an in-solution synthesis to deliver caged pLys peptides (Figure 7A) or a strategy employing base-labile resins, thereby granting direct access to free pLys peptides from cyano ethyl-protected phosphoramidates with only one purification step (Figure 7B).16,61 Peptides obtained via this synthetic approach were, for example, applied in hydrolysis experiments revealing stability high enough for proteomic approaches (t1/2, 25 °C, pH 7.4 ≈ 24 h).

phosphonic acid (2-pAIE) was used as an effective pArg analogue for antibody generation, demonstrating a negligible influence of the chain length over the amidine moiety.28 However, their cross-reactivity studies did not include other easily missed phosphoamino acids. Synthesis of pLys Peptides and Analogues

There are several studies indicating the occurrence of pLys in mammalian cells.38 Zetterqvist and co-workers were the first to report on pLys on a high weight protein isolated from lysate.31 Later, the Smith group revealed the occurrence of pLys in regenerating rat liver and Walker-256 carcinoma.58 Further studies by the Kumon group identified an inorganic pyrophosphate phosphatase as putative pLys phosphatase, although no activity study on Lys-monophosphorylated peptides were reported.59 Compared to pHis and pArg, no specific modification site or specific kinase to install pLys has yet been determined. 1887


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Figure 8. (A) Chemoselective synthesis of pCys peptides and proteins with a two-step approach66 and (B) chemoselective synthesis route to enantiopure pCys peptides.17

Figure 9. (A) Chemoselective synthesis route to site-specifically pyrophosphorylated Ser18 and Thr70 peptides and (B) stable ppSer analogue suited for SPPS.72

Phosphorothioate: Synthesis of pCys Peptides and Analogues

Synthesis of ppSer and ppThr Peptides and Analogues

Another class of anhydrides, pyrophosphorylated proteins, have recently caught the attention of several research groups as nonenzymatic PTM like demonstrated in vitro.68,69 To date, the biological importance of protein pyrophosphorylation is still uncertain. To understand the regulation, metabolism, and biological effect of pyrophosphorylation, the Fiedler group has established a synthesis route for site-specifically pyrophosphorylated Ser18 and Thr70 peptides. By taking advantage of the distinct reactivity of pSer or pThr residues, they could install the pyrophosphate bond by means of a phosphorimidazolide reagent. Subsequent hydrogenic deprotection resulted in the desired pyrophosphorylated peptides (Figure 9A). With such peptides in hand, hydrolytic stability was examined (t1/2, 25 °C, pH7.2 ≫ 24 h),18 and very recently, an applicable MS/MS approach was developed as described later.71 Apart from the synthesis of native ppSer or ppThr peptides, the Fiedler group also developed an Fmoc/Benzyl-protected bisphosphonate as a stable analogue that could be incorporated into peptides during SPPS (Figure 9B).72 With such synthetic peptides in hand, the incorporation in proteins via NCL and also the coupling to maleimide-bovine serum albumin could be demonstrated.

Phospho-Cys is referred to as key amino acid in the active site of different phosphatases, e.g., PTB1B or phosphatases of regenerating liver.62,63 Moreover, it was shown to occur in the phosphoenolpyruvate-dependent phosphotransferase system within the glycosylis pathway of Escherichia coli17 and was eventually identified to play a key role in the bacterial resistance of Staphylococcus aureus mediated by SarA/MgrA proteins.64 The first synthetic reports to install pCys residues as mimics of pSer in peptides and proteins were contributed by Davis and co-workers by a two-step synthetic protocol.65,66 Cysteine was eliminated in proteins to a dehydroalanine residue before pCys was generated by thiophosphate addition as L- and D-isomers (Figure 8A).66 In a recent study, Brik and co-workers investigated the selectivity of the conjugate addition to be ∼60:40 for the D- and L-form independent of the size of the reacted nucleophile.67 Very recently, the Hackenberger group developed a chemoselective route to pCys-containing peptides under preservation of the stereochemistry.17 There, transformation of the Cys-thiol with Ellman’s reagent gave access to an electrophilic disulfide, which reacted selectively with various phosphites to the respective phosphorothioates (Figure 8B). 31 P and 1H NMR studies as well as RP-UPLC experiments verified that the L-form is retained. Furthermore, stability studies with generated peptides revealed a half-life of pCys of approximately 28 h at physiological conditions.17 It is important to note that both synthetic strategies tolerate only one accessible Cys and thereby cannot give access to site-specifically phosphorylated samples.

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APPROPRIATE PROTEOMIC STRATEGIES FOR LABILE PHOSPHORYLATIONS MS-based phosphoproteomics has emerged as the core technology to identify and quantify new endogenous phosphorylation events.3 The majority of phosphoproteomic studies rely on bottom-up phosphoproteomic approaches, wherein proteins from lyzed cells are digested using a protease (most commonly trypsin) followed by nano-liquid chromatog1888


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Accounts of Chemical Research Table 1. Overview of Strategies to Study Labile Phosphorylations and Their Results phosphorylation type pHis24,25,74 pArg21,51,84,85 pLys16,82 pCys17,64

ppSer/ ppThr19,71

cell type

# phosphorylation sites/proteins

MS/MS fragmentation technique

antibody antibody TiO2, YwlE phosphatase trap, ClpPtrapping mutant, antibody

E. coli mammalian (HEK 293) B. subtillis

15/14 780 potential protein substrates 217/134

CID HCD CID, HCD, ECD, ETD

TiO2 SDS-PAGE, RP-chromatography

Staphylococcus aureus phosphoenolpyruvate (PEP)-dependent phosphotransfer system E.coli

enrichment/fractionation

dinuclear zinc(II) complex

2/2 1/1

ETD CID CID, EThcD EThcD

sites could be characterized in E. coli.74 Nevertheless, confident pHis site assignment via CID can be misleading because of dominant neutral losses, low fragmentation efficiency, and intermolecular gas-phase phosphate transfers.76,77 For Ophosphorylated peptides, neutral loss can be circumvented by derivatization of the injected samples with a bis-gallium complex to cage the phosphate ester,78 but no such method has yet been described for labile phosphorylations. Similar observations have been made for pArg peptides. Studies of the fragmentation behavior revealed low spectral quality, extensive neutral loss of phosphoric acid, and gas-phase rearrangements to other phospho-acceptors.79 For these limitations to be overcome, alternative electron-driven fragmentation techniques such as electron-capture dissociation (ECD) and electron-transfer dissociation (ETD) have been applied for analysis of labile PTMs.80,81 These techniques allow fragmentation without loss of labile phosphorylations, thus granting access to confident phosphosite localization of pHis and pArg peptides.76,79 On the basis of these results, synthetically derived sitespecifically modified phospholysine peptides (see Figure 7A) were subjected to different MS/MS experiments. Only ETD provided MS/MS spectra, which allowed phosphosite assignment.16 Nevertheless, it was demonstrated by Krause and coworkers that, even under ETD conditions, migrations of the phosphate moiety to other phospho-acceptors cannot be excluded. Charge state and proton mobility of the precursor ion are important factors that have an impact on phosphate scrambling in the analysis of unstable peptide phosphoramidates.82 During the analysis of cysteine phosphorylation of samples obtained as described in Figure 8B, a fragmentation experiment combining electron transfer and higher energy collision dissociation (EThcD) has been shown to result in higher sequence coverage as well as more confident phosphosite assignment compared to those of ETD and HCD.83 EThcD analysis finally revealed an endogenous pCys site in the phosphoenolpyruvate-dependent phosphotransferase system.17 However, EThcD is the method of choice for analyzing labile phosphorylations; it has limitations regarding its longer cycle times and lower sensitivity compared to those of CID or HCD fragmentation. Very recently, Penkert et al. reported a CIDtriggered EThcD approach, overcoming those restrictions.71 For the pyrophosphorylated peptides to be analyzed, CID fragmentation of all precursor ions was performed. Subsequently, but only upon detection of neutral losses of 98 and 178 Da being characteristic for phosphate and pyrophosphate, respectively, an additional EThcD fragmentation of the same precursor ion was performed. This “on-the-fly”

raphy coupled to tandem mass spectrometry (nLC-MS/MS). Phosphorylated peptides often appear in substoichiometric amounts and exhibit reduced ionization efficiency compared to their unmodified counterparts.73 A variety of enrichment procedures using immunoenrichment by phosphospecific antibodies, immobilized metal affinity chromatography (IMAC), and titanium dioxide (TiO2) have been implemented, facilitating the identification of thousands of serine, threonine, and tyrosine phosphosites within a single LC-MS/MS run.3 Nevertheless, because of their intrinsic labilities, common phosphoproteomic enrichment protocols are limited in enriching N- and S-phosphorylated peptides. To manage this deficiency, we pursued different strategies (Table 1). For instance, antibody enrichment using pan-specific pHis antibodies was used to enrich pHis peptides. After immunoprecipitation, pHis peptides were eluted with pTze, desalted using slightly basic conditions, and eventually analyzed via LC-MS/MS (see below).24,74 A completely different strategy to distinguish between N- and O-phosphorylations and identify arginine phosphorylations used a trapping mutant from YwlEG.ste arginine phosphatase with high affinity for pArg.21 YwlE-bound proteins are either eluted by boiling the beads in SDS sample buffer before SDSPAGE analysis or incubation with high salt concentrations. For phosphoproteomic analysis, bound proteins are digested onbead. In combination with a TiO2-based phosphopeptide enrichment protocol optimized for acid-labile phosphoramidates, pArg sites are eventually identified via LC-MS/MS. An affinity reagent for enrichment of pyrophosphorylated peptides was recently introduced by Fiedler et al.19 A dinuclear zinc(II) complex was attached to a solid support and has proven to catch and release ppSer- or ppThr-peptides in the presence of protein mixtures from the lysate. During enrichment, unspecific bound and singly phosphorylated peptides are washed from the resin using inorganic phosphate following elution of pyrophosphopeptides with inorganic pyrophosphate. Besides the stringent requirements for enrichment of peptides carrying a labile phosphorylation, their analysis and unambiguous localization via LC-MS/MS constitutes another great challenge in phosphoproteomics. Because of its high sensitivity and speed, collision-based fragmentation techniques such as collision-induced dissociation (CID) and multistage activation (MSA)75 are prevalent methods to identify Ophosphorylated peptides. A reported proteomic strategy to study pHis utilizes the elimination of phosphoric and phosphorous acid (neutral losses) during CID MS/MS. In combination with a selective immunoenrichment as described above and a software tool, which exploits the neutral loss pattern of 80, 98, and 116 Da, a number of endogenous pHis 1889


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Figure 10. (A) Extracted ion chromatogram (XIC) of isobaric di- and pyrophosphorylated peptides and the corresponding CID MS/MS spectra showing different neutral loss patterns. (B) EThcD MS/MS spectrum of a pyrophosphorylated peptide exhibiting complete sequence coverage without loss of the labile modification, allowing unambiguous pyrophosphosite assignment. (C) After a high resolution survey scan (HRMS), precursor ions are fragmented in a data-dependent manner via CID MS/MS. As soon as neutral losses of 98 and 178 Da occur, an additional EThcD MS/MS experiment of the same precursor is triggered.71

Notes

method allows the difficult distinction between isobaric pyroand diphosphorylated peptides and, consequently, an unambiguous assignment of the modification site (Figure 10).

The authors declare no competing financial interest.

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Biographies

REMAINING CHALLENGES As can be concluded from the studies presented above, research focusing on the biological role for pHis and pArg is noticeably elaborated to date. On the contrary, the functional understanding of Lys, Cys, as well as pSer/pThr phosphorylation is still in its infancy. Even though considerable progress has been achieved regarding the development of new synthetic and MSbased proteomic tools to study such PTMs, researchers are still in need to demonstrate successful application with unknown samples. Associated therewith, further optimizations regarding the site-specific generation of certain phosphorylated amino acids, expansion of current approaches to proteins, the development of stable analogues for pLys and pCys, and, probably most crucially, generation of specific enrichment and antibody-based techniques are desperately sought. Once obtained, these tools would grant access to diverse biochemical applications, e.g., pull-down assays and enzyme activity probes, leading to further insights into the role of these to date poorly understood PTMs.

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Anett Hauser was born in Berlin, Germany. She obtained her B.S. and M.S. in chemistry in 2012 and 2015, respectively, from the HumboldtUniversität zu Berlin. In 2015, she joined Christian Hackenberger’s group at the Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) for her Ph.D. studies, which are funded by the WernerSchwarze fellowship of the Evonik Stiftung. A chemist by training, her research focuses on the elucidation of the biological role of lysine phosphorylation with the help of chemical biology approaches. Martin Penkert is a Ph.D. candidate in the mass spectrometry core facility of Dr. Eberhard Krause and group member of Christian Hackenberger at the Leibniz-Forschungsinstitut für Molekulare Pharmakologie. He received his M.S. in chemistry in 2014 at the Humboldt-Universität zu Berlin. His research focuses on the development of mass spectrometric methods for analyzing labile phosphorylations. Christian P. R. Hackenberger completed his graduate studies at the universities of Freiburg and UW Madison and his doctoral studies in 2003 at the RWTH Aachen. After his postdoctoral stay at MIT he started his own lab at the Freie Universität Berlin in 2005. In 2012, he accepted a call as the Leibniz-Humboldt Professor for Chemical Biology to the Leibniz-Forschungsinstitut für Molekulare Pharmakologie and the Humboldt-Universität zu Berlin. His lab is interested in studying the biological function and pharmacological potency of naturally and unnaturally modified peptides and proteins, in particular by developing new chemoselective and biorthogonal reactions and novel concepts in protein synthesis.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 1890


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ACKNOWLEDGMENTS The authors acknowledge support from the DFG (SFB 765 and SPP 1623), the Evonik Stiftung (Doctoral fellowship to A.H.), the Fonds der Chemischen Industrie, the Einstein Foundation Berlin, and the Boehringer-Ingelheim Foundation (Plus 3 award).

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