The SPOC domain is a phosphoserine binding module that bridges transcription machinery with co- and post-transcriptional regulators.

The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3'end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of m6A, the most abundant RNA modification. RBM15 positively regulates m6A levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes.

A novel RNA pol II CTD interaction site on the mRNA capping enzyme is essential for its allosteric activation.

Recruitment of the mRNA capping enzyme (CE/RNGTT) to the site of transcription is essential for the formation of the 5' mRNA cap, which in turn ensures efficient transcription, splicing, polyadenylation, nuclear export and translation of mRNA in eukaryotic cells. The CE GTase is recruited and activated by the Serine-5 phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. Through the use of molecular dynamics simulations and enhanced sampling techniques, we provide a systematic and detailed characterization of the human CE-CTD interface, describing the effect of the CTD phosphorylation state, length and orientation on this interaction. Our computational analyses identify novel CTD interaction sites on the human CE GTase surface and quantify their relative contributions to CTD binding. We also identify, for the first time, allosteric connections between the CE GTase active site and the CTD binding sites, allowing us to propose a mechanism for allosteric activation. Through binding and activity assays we validate the novel CTD binding sites and show that the CDS2 site is essential for CE GTase activity stimulation. Comparison of the novel sites with cocrystal structures of the CE-CTD complex in different eukaryotic taxa reveals that this interface is considerably more conserved than previous structures have indicated.

Phosphoserine inhibits neighboring arginine methylation in the RKS motif of histone H3.

The effects of phosphorylation of histone H3 at serine 10 have been studied in the context of other posttranslational modifications such as lysine methylation. We set out to investigate the impact of phosphoserine-10 on arginine-8 methylation. We performed methylation reactions using peptides based on histone H3 that contain a phosphorylated serine and compared the extent of arginine methylation with unmodified peptides. Results obtained via fluorography indicate that peptides containing a phosphorylated serine-10 inhibit deposition of methyl groups to arginine-8 residues. To further explore the effects of phosphoserine on neighboring arginine residues, we physically characterized the non-covalent interactions between histone H3 phosphoserine-10 and arginine-8 using 31P NMR spectroscopy. A salt bridge was detected between the negatively charged phosphoserine-10 and the positively charged unmodified arginine-8 residue. This salt bridge was not detected when arginine-8 was symmetrically dimethylated. Finally, molecular simulations not only confirm the presence of a salt bridge but also identify a subset of electrostatic interactions present when arginine is replaced with alanine. Taken together, our work suggests that the negatively charged phosphoserine maximizes its interactions. By limiting its exposure and creating new contacts with neighboring residues, it will inhibit deposition of neighboring methyl groups, not through steric hindrance, but by forming intrapeptide interactions that may mask substrate recognition. Our work provides a mechanistic framework for understanding the role of phosphoserine on nearby amino acid residues and arginine methylation.

Complexation of Injectable Biphasic Calcium Phosphate with Phosphoserine-Presenting Dendrons with Enhanced Osteoregenerative Properties.

Injectable biphasic calcium phosphates have been proposed as a solution in the treatment of a range of clinical applications including as fillers in the augmentation of osteoporotic bone. To date, various biodegradable natural or synthetic organics have been used as a polymer component of bone materials to increase their cohesiveness. Herein, a novel bone material was developed combining osteoconductive biphasic calcium phosphate (BCP) nanoparticles with phosphoserine-tethered generation 3 poly(epsilon-lysine) dendron (G3-K PS), a class of hyperbranched peptides previously shown to induce biomineralization and stem cell osteogenic differentiation. Strontium was also incorporated into the BCP nanocrystals (SrBCP) to prevent bone resorption. Within 24 h, an antiwashout behavior was observed in G3-K PS-integrated pure BCP group (BCPG3). Moreover, both in vitro tests by relevant cell phenotypes and an in vivo tissue regeneration study by an osteoporotic animal bone implantation showed that the integration of G3-K PS would downregulate Cxcl9 gene and protein expressions, thus enhancing bone regeneration measured as bone mineral density, new bone volume ratio, and trabecular microarchitectural parameters. However, no synergistic effect was found when Sr was incorporated into the BCPG3 bone pastes. Notably, results indicated a concomitant reduction of bone regeneration potential assessed as reduced Runx2 and PINP expression when bone resorptive RANKL and CTX-I levels were reduced by Sr supplementation. Altogether, the results suggest the potential of injectable BCPG3 bone materials in the treatment of osteoporotic bone defects.

Multiple Site-Specific Phosphorylation of IDPs Monitored by NMR.

In line with their high accessibility, disordered proteins are exquisite targets of kinases. Eukaryotic organisms use the so-called intrinsically disordered proteins (IDPs) or intrinsically disordered regions of proteins (IDRs) as molecular switches carrying intracellular information tuned by reversible phosphorylation schemes. Solvent-exposed serines and threonines are abundant in IDPs, and, consistently, kinases often modify disordered regions of proteins at multiple sites. In this context, nuclear magnetic resonance (NMR) spectroscopy provides quantitative, residue-specific information that permits mapping of phosphosites and monitoring of their individual kinetics. Hence, NMR monitoring emerges as an in vitro approach, complementary to mass-spectrometry or immuno-blotting, to characterize IDP phosphorylation comprehensively. Here, we describe in detail generic protocols for carrying out NMR monitoring of IDP phosphorylation, and we provide a number of practical insights that improve handiness and reproducibility of this method.

Substrate Activation of the Low-Molecular Weight Protein Tyrosine Phosphatase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis is known to express a low-molecular weight protein tyrosine phosphatase. This enzyme, denoted as MptpA (Mycobacterium protein tyrosine phosphatase A), is essential for the pathogen to escape the host immune system and therefore represents a target for the search of antituberculosis drugs. MptpA was shown to undergo a conformational transition during catalysis, leading to the closure of the active site, which is by this means poised to the chemical step of dephosphorylation. Here we show that MptpA is subjected to substrate activation, triggered by p-nitrophenyl phosphate or by phosphotyrosine. Moreover, we show that the enzyme is also activated by phosphoserine, with serine being ineffective in enhancing MptpA activity. In addition, we performed assays under pre-steady-state conditions, and we show here that substrate activation is kinetically coupled to the closure of the active site. Surprisingly, when phosphotyrosine was used as a substrate, MptpA did not obey Michealis-Menten kinetics, but we observed a sigmoidal dependence of the reaction velocity on substrate concentration, suggesting the presence of an allosteric activating site in MptpA. This site could represent a promising target for the screening of MptpA inhibitors exerting their action independently of the binding to the enzyme active site.

Library Selection with a Randomized Repertoire of (ßα)8-Barrel Enzymes Results in Unexpected Induction of Gene Expression.

The potential of the frequently encountered (ßα)8-barrel fold to acquire new functions was tested by an approach combining random mutagenesis and selection in vivo. For this purpose, the genes encoding 52 different phosphate-binding (ßα)8-barrel proteins were subjected to error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic Escherichia coli strains, each lacking an enzyme with a phosphate-containing substrate. After plating of the different transformants on minimal medium, growth was observed only for two strains, lacking either the gene for the serine phosphatase SerB or the phosphoserine aminotransferase SerC. The same mutants of the E. coli genes nanE (encoding a putative N-acetylmannosamine-6-phosphate 2-epimerase) and pdxJ (encoding the pyridoxine 5'-phosphate synthase) were responsible for rescuing both ΔserB and ΔserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or SerC reactions in vitro. Instead, RT-qPCR, RNAseq, and transcriptome analysis showed that they rescue the deletions by enlisting the help of endogenous E. coli enzymes HisB and HisC through exclusive up-regulation of histidine operon transcription. While the promiscuous SerB activity of HisB is well-established, our data indicate that HisC is promiscuous for the SerC reaction, as well. The successful rescue of ΔserB and ΔserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (ßα)8-barrel fold.

Serine-Phosphorylated STAT3 Promotes Tumorigenesis via Modulation of RNA Polymerase Transcriptional Activity.

Deregulated activation of the latent oncogenic transcription factor STAT3 in many human epithelial malignancies, including gastric cancer, has invariably been associated with its canonical tyrosine phosphorylation and enhanced transcriptional activity. By contrast, serine phosphorylation (pS) of STAT3 can augment its nuclear transcriptional activity and promote essential mitochondrial functions, yet the role of pS-STAT3 among epithelial cancers is ill-defined. Here, we reveal that genetic ablation of pS-STAT3 in the gp130 F/F spontaneous gastric cancer mouse model and human gastric cancer cell line xenografts abrogated tumor growth that coincided with reduced proliferative potential of the tumor epithelium. Microarray gene expression profiling demonstrated that the suppressed gastric tumorigenesis in pS-STAT3-deficient gp130 F/F mice associated with reduced transcriptional activity of STAT3-regulated gene networks implicated in cell proliferation and migration, inflammation, and angiogenesis, but not mitochondrial function or metabolism. Notably, the protumorigenic activity of pS-STAT3 aligned with its capacity to primarily augment RNA polymerase II-mediated transcriptional elongation, but not initiation, of STAT3 target genes. Furthermore, by using a combinatorial in vitro and in vivo proteomics approach based on the rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) assay, we identified RuvB-like AAA ATPase 1 (RUVBL1/Pontin) and enhancer of rudimentary homolog (ERH) as interacting partners of pS-STAT3 that are pivotal for its transcriptional activity on STAT3 target genes. Collectively, these findings uncover a hitherto unknown transcriptional role and obligate requirement for pS-STAT3 in gastric cancer that could be extrapolated to other STAT3-driven cancers. SIGNIFICANCE: These findings reveal a new transcriptional role and mandatory requirement for constitutive STAT3 serine phosphorylation in gastric cancer.

Growing a Nucleotide/Lanthanide Coordination Polymer Shell on Liposomes.

Coating liposomes with a shell is a useful strategy to increase membrane stability and prevent leakage or fusion. Nucleotide/lanthanide coordination nanoparticles (NPs) are formed by a simple mixing at ambient conditions. Because some lipid headgroups contain lanthanide binding ligands, they may direct the growth of such coordination NPs. Herein, a gadolinium/adenosine monophosphate (Gd3+/AMP) shell was formed on liposomes (liposome@Gd3+/AMP) using lipids containing phosphoserine (PS) or cholinephosphate (CP) headgroups, while phosphocholine liposomes did not support the shell. Liposome binding Gd3+ is confirmed by transmission electron microscopy (TEM). The negatively charged CP and PS liposomes reversed to positive upon Gd3+ binding, while other metals such as Ca2+ and Zn2+ did not reverse the charge. Binding of Gd3+ did not leak the PS liposomes. Then, AMP was further added to cross-link Gd3+ on the liposome surface. A shell was formed as indicated by TEM, and the content inside the liposome remained for the PS liposomes. While adding Triton X-100 still induced leakage of the encapsulated liposomes, the shell protected the liposomes from leakage induced by ZnO NPs, suggesting a porous structure of the Gd3+/AMP shell which allowed penetration of Triton X-100 but not the larger ZnO NPs. This work provides a simple method to coat liposomes, and also offers a fundamental understanding of liposome adsorption of lanthanide ions.

Crystal structures and snapshots along the reaction pathway of human phosphoserine phosphatase.

The equilibrium between phosphorylation and dephosphorylation is one of the most important processes that takes place in living cells. Human phosphoserine phosphatase (hPSP) is a key enzyme in the production of serine by the dephosphorylation of phospho-L-serine. It is directly involved in the biosynthesis of other important metabolites such as glycine and D-serine (a neuromodulator). hPSP is involved in the survival mechanism of cancer cells and has recently been found to be an essential biomarker. Here, three new high-resolution crystal structures of hPSP (1.5-2.0 Å) in complexes with phosphoserine and with serine, which are the substrate and the product of the reaction, respectively, and in complex with a noncleavable substrate analogue (homocysteic acid) are presented. New types of interactions take place between the enzyme and its ligands. Moreover, the loop involved in the open/closed state of the enzyme is fully refined in a totally unfolded conformation. This loop is further studied through molecular-dynamics simulations. Finally, all of these analyses allow a more complete reaction mechanism for this enzyme to be proposed which is consistent with previous publications on the subject.

Anomalous inter-membrane cholesterol transport in fluid phase phosphoserine vesicles driven by headgroup ordered to disordered entropic transition.

POPS is highly enriched in the inner leaflet of the plasma membrane. Here we present measurements of inter-membrane cholesterol transport rates in POPS vesicles. We find that the cholesterol transport kinetics are not only an order of magnitude slower than in POPC lipids at near physiological temperatures, they exhibit a surprising discontinuous Arrhenius behavior around 48 °C. Moreover, thermodynamic analysis suggests that for biologically relevant temperatures, below the discontinuity, the exchange of cholesterol is entropically dominated while it is enthalpically driven, as is the case in POPC vesicles, above that discontinuity. Using the polar fluorescent probe Laurdan we found that POPS fluid membranes retain a large degree of order in the headgroup region for temperatures below the discontinuity but undergo an order-to-disorder transition in the region coinciding with the discontinuity in the transport of cholesterol in POPS membranes providing an explanation not only for the discontinuity but for the entropic dominance at physiological temperatures.

Negative feedback regulation of ErbB4 tyrosine kinase activity by ERK-mediated non-canonical phosphorylation.

ErbB4 receptor tyrosine kinase has four different isoforms that are classified based on variants in the extracellular juxtamembrane domain (JM-a and JM-b) and the C-terminal region (CYT-1 and CYT-2). Here, we used the JM-b/CYT-1 isoform to investigate the roles of serine/threonine phosphorylation in MEK-ERK-dependent feedback inhibition. TPA as an activator of the ERK pathway markedly induced ErbB4 phosphorylation at Thr-674, the conserved common feedback site in the intracellular JM domain, which resulted in the downregulation of tyrosine autophosphorylation. We also identified Ser-1026 as an ErbB4-specific ERK target site in the CYT-1 region. Moreover, double mutations (Thr-674/Ser-1026 to Ala) significantly upregulated ErbB4 activation, indicating that Thr-674 and Ser-1026 are cooperatively involved in negative feedback regulation. Given the fact that ErbB4 mutation is one of the most common genetic alterations in melanoma cells, we demonstrated that a typical oncogenic ErbB4 mutant was resistant to the negative feedback regulation to maintain a highly active status of tyrosine kinase activity. Together, these findings indicate that feedback mechanisms are key switches determining oncogenic potentials of ErbB receptor kinases.

Development of Highly Selective 1,2,3-Triazole-containing Peptidic Polo-like Kinase 1 Polo-box Domain-binding Inhibitors.

Members of the polo-like kinase (Plk) family of serine/threonine protein kinases play crucial roles in cell cycle regulation and proliferation. Of the five Plks (Plk1-5), Plk1 is recognized as an anticancer drug target. Plk1 contains multiple structural components that are important for its proper biological function. These include an N-terminal catalytic domain and a C-terminal non-catalytic polo-box domain (PBD). The PBD binds to phosphothreonine (pT) and phosphoserine-containing sequences. Blocking PBD-dependent interactions offers a potential means of down-regulating Plk1 function that is distinct from targeting its ATP-binding site. Previously, we demonstrated by tethering alkylphenyl chains from the N(π)-position of the His residue in the 5-mer PLHSpT, that we were able to access a hydrophobic "cryptic" binding pocket on the surface of the PBD, and in so doing enhance binding affinities by approximately 1000-fold. More recently, we optimized these PBD-ligand interactions using an oxime ligation-based strategy. Herein, using azide-alkyne cycloaddition reactions, we explore new triazole-containing PBD-binding antagonists. Some of these ligands retain the high PBD-binding affinity of the parent peptide, while showing desirable enhanced selectivity for the PBD of Plk1 relative to the PBDs of Plk2 and Plk3.

Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo.

The posttranslational modification of histones is crucial in spermatogenesis, as in other tissues; however, during spermiogenesis, histones are replaced with protamines, which are critical for the tight packaging of the DNA in sperm cells. Protamines are also posttranslationally modified by phosphorylation and dephosphorylation, which prompted our investigation of the underlying mechanisms and biological consequences of their regulation. On the basis of a screen that implicated the heat shock protein Hspa4l in spermatogenesis, we generated mice deficient in Hspa4l (Hspa4l-null mice), which showed male infertility and the malformation of sperm heads. These phenotypes are similar to those of Ppp1cc-deficient mice, and we found that the amount of a testis- and sperm-specific isoform of the Ppp1cc phosphatase (Ppp1cc2) in the chromatin-binding fraction was substantially less in Hspa4l-null spermatozoa than that in those of wild-type mice. We further showed that Ppp1cc2 was a substrate of the chaperones Hsc70 and Hsp70 and that Hspa4l enhanced the release of Ppp1cc2 from these complexes, enabling the freed Ppp1cc2 to localize to chromatin. Pull-down and in vitro phosphatase assays suggested the dephosphorylation of protamine 2 at serine 56 (Prm2 Ser56) by Ppp1cc2. To confirm the biological importance of Prm2 Ser56 dephosphorylation, we mutated Ser56 to alanine in Prm2 (Prm2 S56A). Introduction of this mutation to Hspa4l-null mice (Hspa4l -/-; Prm2 S56A/S56A) restored the malformation of sperm heads and the infertility of Hspa4l -/- mice. The dephosphorylation signal to eliminate phosphate was crucial, and these results unveiled the mechanism and biological relevance of the dephosphorylation of Prm2 for sperm maturation in vivo.

Does phosphorylation increase the binding affinity of aluminum? A computational study on the aluminum interaction with serine and O-phosphoserine.

Several toxic effects arise from aluminum's presence in living systems, one of these effects is to alter the natural role of enzymes and non-enzyme proteins. Aluminum promotes the hyperphosphorylation of normal proteins. In order to assess the aluminum-binding abilities of phosphorylated proteins and peptides, the interaction of aluminum at different pH with serine and phosphoserine is studied by a Density Functional Theory study, combined with polarizable continuum models to account for bulk solvent effects, and the electronic structure of selected complexes are analyzed by Quantum Theory of "Atoms in Molecules". Our results confirm the high ability of aluminum to bind polypeptides as the pH lowers. Moreover, the phosphorylation of the building blocks increases the affinity for aluminum, in particular at physiological pH. Finally, aluminum shows a tendency to be chelated forming different size rings.

Phosphoserine acidic cluster motifs bind distinct basic regions on the µ subunits of clathrin adaptor protein complexes.

Protein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a potential PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro The sites of these serines vary among mammals in a manner suggesting host-pathogen conflict, yet the Serinc3 PSAC seems dispensable for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu and furin and the PSAC-containing loop of Serinc3 each bind the µ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol 4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

Bioinspired Mineral-Organic Bioresorbable Bone Adhesive.

Bioresorbable bone adhesives have potential to revolutionize the clinical treatment of the human skeletal system, ranging from the fixation and osteointegration of permanent implants to the direct healing and fusion of bones without permanent fixation hardware. Despite an unmet need, there are currently no bone adhesives in clinical use that provide a strong enough bond to wet bone while possessing good osteointegration and bioresorbability. Inspired by the sandcastle worm that creates a protective tubular shell around its body using a proteinaceous adhesive, a novel bone adhesive is introduced, based on tetracalcium phosphate and phosphoserine, that cures in minutes in an aqueous environment and provides high bone-to-bone adhesive strength. The new material is measured to be 10 times more adhesive than bioresorbable calcium phosphate cement and 7.5 times more adhesive than non-resorbable poly(methyl methacrylate) bone cement, both of which are standard of care in the clinic today. The bone adhesive also demonstrates chemical adhesion to titanium approximately twice that of its adhesion to bone, unlocking the potential for adherence to metallic implants during surrounding bony incorporation. Finally, the bone adhesive is shown to demonstrate osteointegration and bioresorbability over a 52-week period in a critically sized distal femur defect in rabbits.

Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions.

Post-translational phosphorylation is essential to human cellular processes, but the transient, heterogeneous nature of this modification complicates its study in native systems. We developed an approach to interrogate phosphorylation and its role in protein-protein interactions on a proteome-wide scale. We genetically encoded phosphoserine in recoded E. coli and generated a peptide-based heterologous representation of the human serine phosphoproteome. We designed a single-plasmid library encoding >100,000 human phosphopeptides and confirmed the site-specific incorporation of phosphoserine in >36,000 of these peptides. We then integrated our phosphopeptide library into an approach known as Hi-P to enable proteome-level screens for serine-phosphorylation-dependent human protein interactions. Using Hi-P, we found hundreds of known and potentially new phosphoserine-dependent interactors with 14-3-3 proteins and WW domains. These phosphosites retained important binding characteristics of the native human phosphoproteome, as determined by motif analysis and pull-downs using full-length phosphoproteins. This technology can be used to interrogate user-defined phosphoproteomes in any organism, tissue, or disease of interest.

Neutralizing the Detrimental Effect of an N-Hydroxysuccinimide Quenching Reagent on Phosphopeptide in Quantitative Proteomics.

One of the most common chemistries used to label primary amines utilizes N-hydroxysuccinimide (NHS), which is also structurally incorporated in various quantitative proteomic reagents such as isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tags (TMT). In this paper we report detrimental effect of hydroxylamine, a widely used quenching reagent for excess NHS, on phosphopeptides. We found an impairment in the degree of phosphopeptide identification when hydroxylamine-quenched TMT-labeled samples were vacuum-dried and desalted compared to the nondried (just diluted) and desalted ones prior to phosphoenrichment. We have also demonstrated that vacuum-drying in the presence of hydroxylamine promotes ß-elimination of phosphate groups from phosphoserine and phosphothreonine while having a minimalistic effect on phosphotyrosine. Additionally, we herein report that this negative impact of hydroxylamine could be minimized by direct desalting after appropriate dilution of quenched samples. We also found a 1.6-fold increase in the number of phosphopeptide identifications after employing our optimized method. The above method was also successfully applied to human tumor tissues to quantify over 15000 phosphopeptides from 3 mg TMT 6-plex labeled-peptides.