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This study introduces an innovative approach for the real-time and efficient detection of alkaline phosphatase (ALP) activity, using a calcein fluorescence probe and leveraging the static quenching properties of calcein fluorescence by Ce3+ metal ions. In this method, calcein serves as the signal element, with its fluorescence effectively preserved through energy transfer or charge transfer when coordinated with Ce3+. Conversely, ALP catalyzes the phosphopeptide substrate to generate a substantial amount of Pi, preventing calcein fluorescence quenching due to the higher affinity between Pi and Ce3+ compared with that between calcein and Ce3+. The fluorescence intensity ratio (F-F0/F0) exhibited excellent linearity, facilitating sensitive ALP detection. The proposed ALP detection method covers a range from 0 to 1.4 mU/mL (R2 = 0.9942), with the limit of detection at 0.069 mU/mL (S/N = 3). Additionally, this method was successfully applied for detecting ALP in serum samples and studying its inhibitors. This research introduces a novel clinical diagnosis approach for ALP sensing while broadening the potential applications of calcein. [ABSTRACT FROM AUTHOR]

A uniform dispersion of TiO2/SiO2 hollow nanosphere of 290 nm with a high surface area of more than 100 m2g− 1 is prepared using polystyrene spheres as templates after calcination at 600 °C. The TiO2/SiO2 hollow nanospheres are characterized by scanning electron microscopy, transmission electron microscopy, dynamic light scattering, nitrogen adsorption-desorption isotherms, and X-ray diffraction. The TiO2/SiO2 hollow nanospheres are shown to be highly effective in selectively enriching phosphopeptides, resulting in a significant reduction of non-phosphorylated species and an enhanced presence of modified peptides. The favorable results obtained in the present study for the enrichment of phosphoprotein β-casein pave the way toward a similar treatment of other types of biomolecules. [ABSTRACT FROM AUTHOR]

Abnormally hyperphosphorylated tau can be recognized by a variety of phosphoprotein‐binding domains (PBDs) to elicit downstream tau signaling in neuropathology, which has been found to have a potential association with subarachnoid hemorrhage. In this study, the genome‐wide binding behavior of tau phosphorylation sites (p‐sites) to PBDs involved in subarachnoid hyperphosphorylation events was systematically profiled at molecular level by integrating peptide docking, structural minimization, affinity scoring, and binding assay, from which a number of potent PBD–p‐site interaction pairs were identified. It was revealed that the PBD domains exhibit distinct binding preferences for phosphotyrosine, phosphoserine, and phosphothreonine p‐sites; the PBD‐recognition specificity of different tau p‐sites is not overlapped with each other, and their phosphorylations would therefore regulate varying biological functions in tau signaling. A number of PBD–p‐site pairs were identified to have potent binding potency as compared to others. The KCIP–pS[393–399] pair was found as a strong binder, which was further optimized with a rational peptide design protocol to derive a number of affinity‐improved phosphopeptides. Structural analysis revealed diverse noncovalent chemical forces across the complex interface of KCIP domain with a designed high‐affinity pS[393–399]‐d4, which confers both stability and specificity to the domain–peptide complex system, with affinity improved by 10.9‐fold relative to the native pS[393–399]. [ABSTRACT FROM AUTHOR]

The study of the molecular mechanisms of the pathogenesis of Alzheimer's disease (AD) is extremely important for identifying potential therapeutic targets as well as early markers. In this regard, the study of the role of post-translational modifications (PTMs) of β-amyloid (Aβ) peptides is of particular relevance. Serine-8 phosphorylated forms (pSer8-Aβ) have been shown to have an increased aggregation capacity and may reflect the severity of amyloidosis. Here, an approach for quantitative assessment of pSer8-Aβ based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is proposed. The relative fraction of pSer8-Aβ was estimated in the total Aβ-pool with a detection limit of 1 fmol for pSer8-Aβ (1–16) and an accuracy of 2% for measurements in the reflectron mode. The sensitivity of the developed method is suitable for determining the proportion of phosphorylated peptides in biological samples. [ABSTRACT FROM AUTHOR]

For phosphoproteomics, it is important to separate and enrich phosphoproteins from complex biological samples predominantly containing their nonphosphorylated counterparts. This review describes effective chromatographic techniques for the separation and enrichment of phosphoproteins using a unique functional molecule, Phos-tag, which can capture a phosphate monoester dianion in an aqueous solution at neutral pH, developed by mimicking the active center of alkaline phosphatase. Phosphate affinity chromatographic Phos-tag agarose and Phos-tag magnetic beads can be used for the comprehensive enrichment of phosphoproteins under near-physiological conditions, providing information on the nature of native phosphoproteins in cells. The simple procedure using the Phos-tag beads facilitates the efficient enrichment of rare phosphoproteins from complex mixtures, resulting in increased sensitivity of downstream assays. These affinity beads can also be used for the enrichment of phosphopeptides in phosphorylation analysis using mass spectrometry. Phosphopeptide enrichment must be employed to decrease the complexity of the proteome, for which several strategies have been proposed. We introduce the performance of Phos-tag beads compared with other conventional strategies proposed for phosphopeptide enrichment and the impact of Phos-tag-based affinity chromatography for phosphoproteomics.

Collisionally activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), electron capture dissociation and electron detachment dissociation (EDD) experiments were conducted on a set of phosphopeptides, in a Fourier transform ion cyclotron resonance mass spectrometer. The fragmentation patterns were compared and varied according to the fragmentation mechanisms and the composition of the peptides. CAD and IRMPD produced similar fragmentation profiles of the phosphopeptides, while UVPD produced a large number of complementary fragments. Electron-based dissociation techniques displayed lower fragmentation efficiencies, despite retaining the labile phosphate group, and drastically different fragmentation profiles. EDD produced complex spectra whose interpretation proved challenging. [ABSTRACT FROM AUTHOR]

Keywords: basophils; casein; cow milk; food allergy; milk hypersensitivity; phosphopeptide EN basophils casein cow milk food allergy milk hypersensitivity phosphopeptide 1 5 5 03/29/22 20220301 NES 220301 ACKNOWLEDGMENTS We thank all the physicians, nutritionists, and nurses who performed the OFCs and data collection and Mr. Noboru Hayakawa (Clinical Laboratory technician in Aichi Children's Health and Medical Center) and Ms. Kumiko Matsuo (Cell Biology Section, BML, Inc.) for their excellent technical assistance. First, symptoms that developed after ingesting CPP-ACP containing paste or gum were possibly not induced by CPP-ACP because OFC using CPP-ACP was not performed, the time between OFC and episode was sometimes long, and I in vitro i tests were performed with CPP instead of CPP-ACP. We recruited six patients with CMA whose severity was similar to that of CPP-ACP (reacted <10 ml of CM) and who presented with casein-specific IgE titer levels above two digits and completely avoided CM for the in vitro tests (Table S1). Third, the CPP reagent used in our experiments was a mixture of trypsin-digested casein peptides, which might contain residual protein IgE epitopes, along with the CPP region in casein. [Extracted from the article]

The study of the molecular mechanisms of the pathogenesis of Alzheimer’s disease (AD) is extremely important for identifying potential therapeutic targets as well as early markers. In this regard, the study of the role of post-translational modifications (PTMs) of β-amyloid (Aβ) peptides is of particular relevance. Serine-8 phosphorylated forms (pSer8-Aβ) have been shown to have an increased aggregation capacity and may reflect the severity of amyloidosis. Here, an approach for quantitative assessment of pSer8-Aβ based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is proposed. The relative fraction of pSer8-Aβ was estimated in the total Aβ-pool with a detection limit of 1 fmol for pSer8-Aβ (1–16) and an accuracy of 2% for measurements in the reflectron mode. The sensitivity of the developed method is suitable for determining the proportion of phosphorylated peptides in biological samples.

Protein phosphorylation plays an important role in cellular signaling and disease development. Advances in mass spectrometry-based proteomics have enabled qualitative and quantitative phosphorylation studies as well as in-depth biological explorations for biomarker discovery and signaling pathway analysis. However, the dynamic changes that occur during phosphorylation and the low abundance of target analytes render direct analysis difficult because mass spectral detection offers no selectivity, unlike immunoassays such as Western blot and enzyme-linked immunosorbent assay (ELISA). The present study aimed to solve one of the key problems in the specific and efficient isolation of phosphorylated peptides. A method based on a magnetic carbon nitride composite coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed for the enrichment and analysis of phosphopeptides with low abundance in complex samples. Magnetic carbon nitride composite was synthesized and characterized by electron microscopy, infrared spectroscopy, and X-ray diffractometry. The composite showed a well-distributed two-dimensional layered structure and functional groups with excellent paramagnetic performance. Two classical phosphoproteins, namely, α - and β -caseins, were selected as model phosphorylated samples to assess the performance of the proposed enrichment technique. The magnetic carbon nitride composite exhibited high selectivity and sensitivity for phosphopeptide enrichment. The limit of detection was determined by MALDI-TOF-MS analysis to be 0.1 fmol. The selectivity of the method was investigated using the digest mixtures of α -casein, β -casein, and bovine serum albumin (BSA) with different mass ratios (1∶1∶1000, 1∶1∶2000, and 1∶1∶5000). Direct analysis of the samples revealed the dominance of spectral signals from the abundant peptides in BSA. After enrichment with the magnetic carbon nitride composite, the high concentration of background proteins was washed away and only the signals of the phosphopeptides were captured. The signals from the casein proteins were clearly observed with little background noise, indicating the high selectivity of the composite material. The robustness of the method was tested by assessing the reusability of the same batch of magnetic carbon nitride materials over 20 cycles of enrichment. The composite showed nearly the same enrichment ability even after several cycles of reuse, demonstrating its potential applicability for a large number of clinical samples. Finally, the method was applied to the analysis of phosphopeptides from several commonly used phosphoprotein-containing samples, including skimmed milk digest, human serum, and human saliva; these samples are significant in the analysis of food quality, disease biomarkers, and liquid biopsies for cancer. Without enrichment, no phosphopeptide was detected because of the high abundance of nonphosphopeptide materials dominating the spectral signals obtained. After pretreatment with the developed magnetic carbon nitride composite, most of the phosphosites were identified with high selectivity and sensitivity via MALDI-TOF-MS. These results revealed the practicality of the developed approach for clinical applications. In addition, our method may potentially be employed for phosphoproteomics with real complex biological samples.

SUMMARY: Phosphoglycerate mutases (PGAMs) catalyse the reversible isomerisation of 3‐phosphoglycerate and 2‐phosphoglycerate, a step of glycolysis. PGAMs can be sub‐divided into 2,3‐bisphosphoglycerate‐dependent (dPGAM) and ‐independent (iPGAM) enzymes. In plants, phosphoglycerate isomerisation is carried out by cytosolic iPGAM. Despite its crucial role in catabolism, little is known about post‐translational modifications of plant iPGAM. In Arabidopsis thaliana, phosphoproteomics analyses have previously identified an iPGAM phosphopeptide where serine 82 is phosphorylated. Here, we show that this phosphopeptide is less abundant in dark‐adapted compared to illuminated Arabidopsis leaves. In silico comparison of iPGAM protein sequences and 3D structural modelling of AtiPGAM2 based on non‐plant iPGAM enzymes suggest a role for phosphorylated serine in the catalytic reaction mechanism. This is confirmed by the activity (or the lack thereof) of mutated recombinant Arabidopsis iPGAM2 forms, affected in different steps of the reaction mechanism. We thus propose that the occurrence of the S82‐phosphopeptide reflects iPGAM2 steady‐state catalysis. Based on this assumption, the metabolic consequences of a higher iPGAM activity in illuminated versus darkened leaves are discussed. Significance Statement: Serine 82 phosphorylation and dephosphorylation of Arabidopsis thaliana 2,3‐bisphosphoglycerate‐independent phosphoglycerate mutase 2 are crucial steps of the catalytic mechanism. Under the assumption that S82‐phosphopeptide content reflects steady‐state catalysis, in planta leaf iPGAM activity is higher in the light than in the dark. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is a crucial posttranslational modification for the regulation of many different biological functions. Selective enrichment of phosphopeptides from the complex biological samples is an essential step for the mass spectrometry analysis of protein phosphorylation. In this study, an arsenate functionalized monolithic column was first prepared by a single‐step copolymerization of p‐methacryloylaminophenylarsonic acid and ethylene dimethacrylate. Then the metal ions Zr4+ were attached onto the prepared monolithic column via metal‐chelate complex formation by Zr4+ and arsenate groups. The obtained monolithic column was employed as a new sorbent for the phosphopeptide enrichment via immobilized metal affinity chromatography. Phosphopeptides analysis was realized by polymer monolith microextraction using this monolithic column coupled to both matrix‐assisted laser desorption/ionization mass spectrometry and liquid chromatography–electrospray ionization tandem mass spectrometry. The proposed method exhibited a high selectivity for phosphopeptide enrichment in complex matrices, and was applied to the analysis of phosphopeptides in human serum and tryptic digests of rat brain proteins. Four phosphopeptides could be selectively captured from human serum and 2608 endogenous phosphopeptides were identified from the tryptic digests of rat brain proteins, indicating a satisfactory performance of this method for the enrichment of phosphopeptides from complex biological samples. [ABSTRACT FROM AUTHOR]

Brassinosteroids (BRs) are vital plant steroid hormones sensed at the cell surface by a membrane signaling complex comprising the receptor kinase BRI1 and a SERK-family co-receptor kinase. Activation of this complex lead to dissociation of the inhibitor protein BKI1 from the receptor and to differential phosphorylation of BZR1/BES1 transcription factors by the glycogen synthase kinase 3 protein BIN2. Many phosphoproteins of the BR signaling pathway, including BRI1, SERKs, BKI1 and BZR1/BES1 can associate with 14-3-3 proteins. In this study, we use quantitative ligand binding assays to define the minimal 14-3-3 binding sites in the N-terminal lobe of the BRI1 kinase domain, in BKI1, and in BZR1 from Arabidopsis thaliana. All three motifs require to be phosphorylated to specifically bind 14-3-3s with mid- to low micromolar affinity. BR signaling components display minimal isoform preference within the 14-3-3 non-ε subgroup. 14-3-3λ and 14-3-3ω isoform complex crystal structures reveal that BKI1 and BZR1 bind as canonical type II 14-3-3 linear motifs. Disruption of key amino acids in the phosphopeptide binding site through mutation impairs the interaction of 14-3-3λ with all three linear motifs. Notably, quadruple loss-of-function mutants from the non-ε group exhibit gain-of-function brassinosteroid signaling phenotypes, suggesting a role for 14-3-3 proteins as overall negative regulators of the BR pathway. Collectively, our work provides further mechanistic and genetic evidence for the regulatory role of 14-3-3 proteins at various stages of the brassinosteroid signaling cascade., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Phosvitin, one of the most noteworthy bioactive components of hen egg yolk, is an amphiphilic protein that stands out with its unique composition and functionality in the food industry and health. Phosvitin consists of 4% of egg yolk dry matter and 11% of egg yolk proteins. It is considered as the most phosphorylated protein with 10% phosphorus. Besides, some potential novel phosphopeptides containing clusters of phosphoserines can be derived from hen's egg yolk phosvitin. Phosvitin, which has many functional features thanks to its unique structure, is known primarily for its metal bonds binding (iron, calcium, etc.) feature. On the other hand, its phosphopeptides may increase the bioavailability of metals compared to phosvitin. Although this feature of phosvitin may partially decrease the bioavailability of especially iron in the egg, it allows the phosvitin to have many bioactivities in the food industry and health. Lipid oxidation, which is a serious problem in the food industry, can be inhibited by adding phosvitin and its derived phosphopeptides to the food production chain via inhibiting bivalent iron. Because phosvitin is an amphiphilic protein capable of chelating, it also shows potential antibacterial effects against the Gram‐negative bacteria. Moreover, the literature has recently been attempting to define the promising relationship between phosvitin and its phosphopeptides and plenty of health‐promoting activities such as immune‐enhancing, melanogenesis inhibitor, anti‐ageing, and anticancer. In this review, current information on the hen's egg yolk phosvitin and its phosphopeptides and their bioactivities in the food industry and health are discussed and some future directions are given. [ABSTRACT FROM AUTHOR]

Significant technical advancements in phosphopeptide enrichment have enabled the identification of thousands of p-peptides (mono and multiply phosphorylated) in a single experiment. However, it is still not possible to enrich all p-peptide species in a single step. A range of new techniques and materials has been developed, with the potential to provide a step-change in phosphopeptide enrichment. The first half of this review contains a tutorial for new potential phosphoproteomic researchers; discussing the key steps of a typical phosphoproteomic experiment used to investigate canonical phosphorylation sites (serine, threonine and tyrosine). The latter half then show-cases the latest developments in p-peptide enrichment including: i) Strategies to mitigate non-specific binding in immobilized metal ion affinity chromatography and metal oxide affinity chromatography protocols; ii) Techniques to separate multiply phosphorylated peptides from monophosphorylated peptides (including canonical from non-canonical phosphorylated peptides), or to simultaneously co-enrich other post-translational modifications; iii) New hybrid materials and methods directed towards enhanced selectivity and efficiency of metal-based enrichment; iv) Novel materials that hold promise for enhanced phosphotyrosine enrichment. A combination of well-understood techniques and materials is much more effective than any technique in isolation; but the field of phosphoproteomics currently requires benchmarking of novel materials against current methodologies to fully evaluate their utility in peptide based proteoform analysis. Image 1 Schematic diagram for p-peptide enrichment strategies. Ti4+ lab-in-syringe polydopamine coated three-dimensional porous graphene aerogel sorbent carrying immobilized titanium (IV) ions (denoted as Ti4+@PDA@GA [ 53 ]), Phos-PAD: p-peptide paper-based analytical devices [ 72 ], graphene oxide-trimethyl-2-methacroyloxyethyl ammonium chloride-titania (GO-META-TiO 2) [ 64 ]. PolyMAC: (polymer-based metal ion affinity capture) [ 86 , 87 ]. Phosphotyrosine antibody cocktails [ 102 ]. Phosphotyrosine-imprinted polymer with TiO 2 [ 100 ]. IMAC-antibody (IMAC with phosphotyrosine antibody) [ 15 ]. High pH and pH step wise elution [ 15 ]. Gradient elution: 100 mM NH 4 HCO 3 (pH 9.2–11.3 step gradient and pH adjust by ammonia [ 16 ]. Ti4+@PDA@GA) Adapted with permission [ 53 ]. Copyright 2018, Springer Nature. Phos-PAD) Adapted with permission [ 72 ]. Copyright 2019, Elsevier. GO-META-TiO 2) Adapted with permission [ 64 ]. Copyright 2019, Elsevier. • A critical review of method development in phosphopeptide enrichment. • Existing methods include IMAC, MOAC and other techniques. • Modifications on working condition/supporting ligand/loading capacity. • Development of novel/hybrid/dual affinity materials. • Benchmarking performance of novel methods against existing methods is required. [ABSTRACT FROM AUTHOR]

Introduction: The goal of this review is to survey proteomics and next generation sequencing (NGS) approaches to discover phosphopeptide neoantigens that can be used as targets for individualized cancer therapy and diagnostics. Neoantigens are peptides or proteins that can be derived from tumor-specific or viral alterations containing mutated sequences including different post-translational modifications that will not be found in normal human cells. These neoantigens can be highly specific for the individual affected tissue. Mass spectrometry combined with NGS allow identifying autoantibodies specific for neoantigens, including neoantigens that contain phosphorylated moieties. Areas covered: We discuss recognition of neoantigens through the application of phosphoproteomics. We focus on phosphoprotein modifications that can be accurately identified with mass spectrometry and can serve as rarely described targets for immunotherapy and diagnosis in cancer and autoimmune diseases. Identification of these phospho-moiety containing neoantigens gives the possibility to search for corresponding autoantibodies, especially if NGS and mass spectrometry are combined. Expert commentary: Identification of post-translational modifications with mass spectrometry gives possibilities to define modifications that are specific for cancer and autoimmune diseases. This technology develops rapidly, and gives opportunities to select neoantigens that are highly specific for cancer and immune diseases and to sequence autoantibodies involved. [ABSTRACT FROM AUTHOR]

Iron(III-immobilized magnetic nano-composites (MNCs) were first fabricated using one-step aqueous self-assembly of oligopeptides (Glu-Pro-Ala-Lys-Ala-Lys-Ala-Lys; EPAK-VI) for the highly selective capture of phosphopeptides from complex biological samples. Under physiological conditions, EPAK-VI can readily self-organize into a robust and complete coating layer mainly composed of β-sheets and β-turns on the surface of Fe3O4@GO and Fe3O4@C MNCs. Tailored by the cyclic structure of proline, the Glu-Pro motifs of EPAK-VI are vertically erected on the surface and thus serve as an effective linker to chelate Fe3+ through carboxyl (COO-) group in the glutamic acid (E) residues. The ionic hydrogen bonds between the ε-amino groups and the surface negative charges coupled with intermolecular hydrogen bonds render the EPAK-VI coating on the MNCs insusceptible to repeated extreme washing conditions. The Fe3+-EPAK-VI coated MNCs exhibit high enrichment efficiency for β-casein tryptic digest (0.05 fmol μL−1), excellent selectivity from mixed digests (β-casein/bovine serum albumin, mass ratio 1:500), and high recovery rate (over 80%). [ABSTRACT FROM AUTHOR]

A hybrid monolith was prepared from perovskite and cucurbit[6]uril [poly(hydroxyethyl methacrylate-pentaerythritol triacrylate) monolith] for the enrichment of phosphopeptides. By coupling with mass spectrometry, three goals were simultaneously realized, viz. (a) selective enrichment of phosphopeptides from non-phosphopeptides, (b) identification of mono- and multi-phosphopeptides, and (c) recognition of tyrosine phosphopeptides. The perovskite introduced into the monolith warrants high selectivity for phosphopeptides even at a high (10,000:1) ratio of non-phosphopeptides to phosphopeptides, and and enables identification of eight mono- and multi-phosphopeptides from standard β-casein tryptic digests. Tyrosine phosphopeptides were specifically detected via the recognition capability of cucurbit[6]uril integrated into the monolith. The method has remarkably specific enrichment capacity for phosphopeptides from samples including human serum, nonfat milk, and human acute myelocytic leukemia cell lysate. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is central to most cellular processes. Despite the importance and widespread occurrence of this modification, phosphoproteome analysis of complex biological samples still remains a challenge. The present study has been undertaken to compare iron affinity immobilized metal ion affinity chromatography (Phos-Select™ IMAC) and Gallium immobilized metal ion affinity chromatography (Ga-IMAC) to enrich and characterize phosphopeptides from the whole-cell lysate of Jurkat cells. Our results indicated that enrichment was dependent on the isoelectric point (pI) and molecular mass of the proteins and it was found to be better in case of PHOS-Select IMAC (51.2%) as compared to Ga-IMAC (28.8%). This study compared and analysed phosphopeptide profile of Jurkat cells by two different IMAC techniques and provides advancement in phosphopeptide enrichment methods.

Antibodies play a crucial role in monitoring post-translational modifications, like phosphorylation, which regulates protein activity and location; however, commercial polyclonal and monoclonal antibodies have limitations in renewability and engineering compared to recombinant affinity reagents. A scaffold based on the Forkhead-associated domain (FHA) has potential as a selective affinity reagent for this post-translational modification. Engineered FHA domains, termed phosphothreonine-binding domains (pTBDs), with limited cross-reactivity were isolated from an M13 bacteriophage display library by affinity selection with phosphopeptides corresponding to human mTOR, Chk2, 53BP1, and Akt1 proteins. To determine the specificity of the representative pTBDs, we focused on binders to the pT543 phosphopeptide (536-IDEDGENpTQIEDTEP-551) of the DNA repair protein 53BP1. ELISA and western blot experiments have demonstrated the pTBDs are specific to phosphothreonine, demonstrating the potential utility of pTBDs for monitoring the phosphorylation of specific threonine residues in clinically relevant human proteins., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Highly efficient enrichment of phosphopeptides in low abundance prior to mass spectrometry analysis is essential in phosphopeptidomics analysis. Herein, an affinity probe possessing both interactions of polyoxometalates (POMs) and hydrophilic interaction chromatography (HILIC) was facilely prepared. POMs and cysteamine hydrochloride-modified chitosan (CYECS) were assembled onto magnetic nanoparticles (MNPs) via a layer-by-layer approach. The developed MNPs-(POM/CYECS) material owned merits of large metal oxide surfaces, positive charge, and hydrophilicity as well as superparamagnetism. By combining with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), the prepared material was utilized for effective enrichment of phosphopeptides. High selectivity (mass ratio of β -casein and bovine serum albumin digests of 1:5000) and low detection limit (0.02 fmol) toward phosphopeptides were achieved. The recovery of phosphopeptides was measured to be 92.6%. The specific enrichment of phosphopeptides from complex samples (nonfat milk, human saliva, serum, and A549 cell lysate) with MNPs-(POM/CYECS) material further confirmed its bright prospects for isolation of phosphopeptides from biological samples. In this study, polyoxometalates and cysteamine hydrochloride-modified chitosan were assembled onto magnetic nanoparticles via a layer-by-layer approach. The developed material was employed as the adsorbent for the selective enrichment of phosphopeptides by coupling with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The material was successfully applied to enrich phosphopetides from standard peptide mixtures, non-fat milk digests, human saliva, serum, and A549 cell lysate. Image 1 • Polyoxometalates and cysteamine hydrochloride-modified chitosan were assembled onto magnetic nanoparticles via a layer-by-layer approach. • The prepared material was applied to enrich phosphopeptides coupled with MALDI-TOF MS determinations. • The prepared material showed efficient enrichment performance with high sensitivity, selectivity, and good reusability for phosphopeptides. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is one of the main mechanisms by which signals are transmitted in eukaryotic cells, and it plays a crucial regulatory role in almost all cellular processes. In yeast, more than half of the proteins are phosphorylated in at least one site, and over 20,000 phosphopeptides have been experimentally verified. However, the functional consequences of these phosphorylation events for most of the identified phosphosites are unknown. A family of protein interaction domains selectively recognises phosphorylated motifs to recruit regulatory proteins and activate signalling pathways. Nine classes of dedicated modules are coded by the yeast genome: 14-3-3, FHA, WD40, BRCT, WW, PBD, and SH2. The recognition specificity relies on a few residues on the target protein and has coevolved with kinase specificity. In the present study, we review the current knowledge concerning yeast phospho-binding domains and their networks. We emphasise the relevance of both positive and negative amino acid selection to orchestrate the highly regulated outcomes of inter- and intra-molecular interactions. Finally, we hypothesise that only a small fraction of yeast phosphorylation events leads to the creation of a docking site on the target molecule, while many have a direct effect on the protein or, as has been proposed, have no function at all. • Reversible protein phosphorylation has a pivotal role in the regulation of the yeast cellular processes. • Phosphoproteomic analyses have identified more than 20,000 phosphorylation sites in yeast. • Interestingly, the majority of these sites have not been functionally characterized. • Seven families of binding domains recognise pS and pT in S. cerevisiae, while pY does not regulate interactions. • The characterization of phospho-binding domains helps to understand the function and the evolution of the phosphoproteome. [ABSTRACT FROM AUTHOR]

Phosphorylation, a major posttranslational modification of proteins, plays an important role in protein activity and cell signaling. However, it is difficult to detect protein phosphorylation because of its low abundance and the fact that the analysis can be hindered by the presence of highly abundant non-phosphoproteins. In order to reduce the sample complexity and improve the efficiency of identification of phosphopeptides, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) was utilized to enrich phosphopeptides from a murine macrophage cell lysate. Strong cation chromatography (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC), and solution isoelectric focusing (sIEF) were investigated in detail for phosphopeptide fractionation strategies followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. A total of 5744 non-redundant phosphopeptides and 2159 phosphoproteins were identified from the cell lysates in three fractionation approaches. The SCX fractionation contained the largest number of phosphoproteins and phosphopeptides that were identified. In addition, 4336, 2064, and 2424 phosphopeptides were identified from SCX-LC-MS/MS, ERLIC-LC-MS/MS, and sIEF-LC/MS-MS, including 2430, 438, and 751 phosphopeptides that were only specifically found in SCX, ERLIC, and sIEF fractionations. In conclusion, these three fractionation strategies demonstrated great complementarity, which greatly improved the efficiency of identification of phosphopeptides and can be suitable for use in in-depth phosphoproteome research. [ABSTRACT FROM AUTHOR]

Abstract It is necessary to effectively absorb trace phosphopeptides/proteins from complex phosphopeptide-containing samples for mass spectrometry (MS)-based phosphoproteomics. Here, Bi3+/Fe3+/Zr4+co-doped TiO 2 (TiO 2 /Bi/Fe/Zr) nanocomposite with a high surface-to-volume ratio was synthesized by sol-gel method for selective enrichment of phosphopeptides. The enrichment ability of this modified TiO 2 toward phosphopeptides was evaluated by using standard phosphoproteins and HeLa cell lysate. The results demonstrated that, compared with commercial titanium dioxide for phosphopeptides enrichment, this proposed TiO 2 /Bi/Fe/Zr nanocomposite has a lower detection limit (4 × 10−10 M) and higher selectivity at a low weight ratio of phosphopeptides/nonphosphopeptides (1:1000). Additionally, a total of 676 phosphorylation sites were identified from the HeLa cell lysate after enrichment by the TiO 2 /Bi/Fe/Zr nanocomposite. In brief, the TiO 2 /Bi/Fe/Zr nanocomposite exhibits better performance on the selectively absorption of phosphopeptides from either simple or complex biological samples, indicating the great potential application in phosphoproteomics study. Graphical abstract fx1 Highlights • It is the first time to prepare TiO 2 /Bi/Fe/Zr nanocomposite for the enrichment of phosphopeptides. What is more, the prepared TiO 2 /Bi/Fe/Zr nanocomposite possess advantages of simple synthesis method and low cost. • The TiO 2 /Bi/Fe/Zr nanocomposite exhibits higher efficiency and specificity in the enrichment of phosphopeptides from either simple or complex phosphopeptide-containing samples than the commercial titanium dioxide.. • The mechanism of the high enrichment efficiency was investigated. [ABSTRACT FROM AUTHOR]

Abstract A novel composite monolithic column based on graphene oxide–trimethyl-2-methacroyloxyethylammonium chloride-titania (GO-META-TiO 2) was developed for the enrichment of phosphopeptides. META was proposed as a "bridge" to connect GO and TiO 2 species to prepare GO-META-TiO 2 composite. This high surface area composite (surface area = 196.93 m2 g−1) was fixed in the monolithic column via an in situ UV polymerization process. In-tube solid phase microextraction (IT-SPME) using this composite was coupled with matrix-assisted laser desorption ionization time-of-flight–mass spectrometry (MALDI-TOF-MS) for the enrichment and detection of phosphorylated peptides from a digestion mixture of α-casein, β-casein, and bovine serum albumin (BSA) in molar ratios of 1:1:1, 1:1:10, and 1:1:100. The key factors affecting the IT-SPME of the phosphopeptides, such as the elution solution concentrations, the extraction flow rate, and the elution flow rate were comprehensively investigated. For further demonstration, this method was employed for the enrichment and detection of phosphorylated peptides from digested chicken egg white. The obtained results indicated that the GO-META-TiO 2 composite monolithic column rapidly and efficiently captured the phosphopeptides present in these complex biological samples, even in the 10 fmol β-casein tryptic digest. We therefore propose that the reported GO-META-TiO 2 composite monolithic column possesses a suitable affinity for the selective extraction of phosphopeptides from biological samples. This method paves a way in extending the application of nanomaterials. Graphical abstract fx1 Highlights • GO-META-TiO 2 composite monolithic columns were developed for the IT-SPME of phosphopeptides. • GO-META-TiO 2 composites were prepared using META as a "bridge" for in situ growth of TiO 2 in the monolithic column. • This method paves a way in extending the application of nanomaterials. [ABSTRACT FROM AUTHOR]

We demonstrate a new, efficient method for the preparation of phosvitin phosphopeptides using immobilised-trypsin enzymolysis technology. Immobilised trypsin was prepared using a covalent binding method, and then was added to degrade egg yolk phosvitin for the production of phosphopeptides. In our results, the prepared immobilised trypsin demonstrated a higher hydrolysing activity toward phosvitin than free trypsin, and the hydrolysing activity was retained well even after trypsin was repeatedly used up to five times. Interestingly, the phosvitin phosphopeptides prepared with immobilised trypsin demonstrated a lower N/P ratio and a higher calcium-binding efficiency than those prepared with free trypsin. Furthermore, phosphopeptides significantly increased the rate of calcium absorption and serum calcium content in vivo. Based on these results, we conclude that trypsin immobilised onto chitosan has a greater phosvitin hydrolysing activity than free trypsin, and the prepared phosphopeptides can be used as a new calcium supplement to significantly increase calcium absorption in growing rats.

We investigated whether diurnal differences in muscle force output are associated with the post-translational state of muscle proteins. Ten physically active men (mean ± SD; age 26.7 ± 3.7 y) performed experimental sessions in the morning (08:00 h) and evening (17:00 h), which were counterbalanced in order of administration and separated by at least 72 h. Knee extensor maximal voluntary isometric contraction (MVIC) force and peak rate of force development (RFD) were measured, and samples of vastus lateralis were collected immediately after exercise. MVIC force was greater in the evening (mean difference of 67 N, 10.2%; p < 0.05). Two-dimensional (2D) gel analysis encompassed 122 proteoforms and discovered 6 significant (p < 0.05; false discovery rate [FDR] = 10%) diurnal differences. Phosphopeptide analysis identified 1693 phosphopeptides and detected 140 phosphopeptides from 104 proteins that were more (p < 0.05, FDR = 22%) phosphorylated in the morning. Myomesin 2, muscle creatine kinase, and the C-terminus of titin exhibited the most robust (FDR < 10%) diurnal differences. Exercise in the morning, compared to the evening, coincided with a greater phosphorylation of M-band-associated proteins in human muscle. These protein modifications may alter the M-band structure and disrupt force transmission, thus potentially explaining the lower force output in the morning.

The enrichment technique is crucial to the comprehensive analysis of protein phosphorylation. In this work, a facile, green and efficient synthetic method was set up for quaternization of luffa sponge. The resultant luffa sponge showed strong anion-exchange characteristics and a high adsorption ability for phosphate ions. Along with the unique physical properties, e.g., tenacity and porous texture, quaternized luffa sponge was demonstrated to be a well-suited solid-phase extraction (SPE) material. The quaternized luffa sponge-based SPE method was simple, cost-effective and convenient in operation, and was successfully applied to the capture of phosphopeptides from protein digests. The enrichment approach exhibited exceptionally high selectivity, sensitivity and strong anti-interference ability. Four phosphopeptides were still detected by using the digest mixture of β-casein and bovine serum albumin with a molar ratio of 1:100. 21 phosphopeptides were identified from the tryptic digest of non-fat milk.

Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564., Competing Interests: Conflict of interest B. C. S. is a founder and shareholder in Proteome Software, which operates in the field of proteomics. A. J. N. and J. M. R. are employees of Cell Signaling Technology. A. W. H. and B. P. are employees of Thermo Fisher Scientific., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The American Academy of Pediatric Dentistry (AAPD) affirms that the use of fluoride, as an adjunct in the prevention of caries, is safe and effective. The AAPD encourages dentists, other healthcare providers, and parents to optimize fluoride exposures to reduce the risk of caries and to enhance the remineralization of affected teeth. However, there is resistance amongst patients towards fluoride overexposure and despite there being research on other effective remineralizing agents, most pediatric dentists primarily cater their practice to fluoride-based products. The objective of the study is to survey pediatric dentists' acceptance and awareness of fluoride-free remineralizing agents. A listserv of the southeastern and western private practice pediatric dentists was obtained from the AAPD consisting of 6490 email addresses. A questionnaire consisting of 15 questions was sent to each address using Qualtrics. Different trends in fluoride-free acceptance and awareness were seen based on region of practice, region of training and age of practitioner. Region of practice, residency training and age can be contributing factors toward fluoride-free remineralizing agent opinion. The data gathered trends towards western-trained pediatric dentists are more likely to recommend a fluoride-free toothpaste than a southeastern-trained dentist., Competing Interests: The authors declare no conflict of interest., (©2023 The Author(s). Published by MRE Press.)

Fe x O y and ZrO 2 nanoparticles co-doped layered porous polyimide, polyimide-Fe x O y -ZrO 2 is prepared with a one-step strategy, shortly termed as PI-Fe x O y -ZrO 2 . The layered and porous structure of the polymer offers a supported platform for metallic oxide anchoring, exhibiting a mesopore size of 3.93 nm and providing a surface area of 198.47 m 2 g −1 . The metallic oxides were uniformly and highly dispersed in the PI-Fe x O y -ZrO 2 nanocomposite with percentages of 15.81 and 20.53 wt% for Fe and Zr, respectively. The magnetic Fe x O y provides driving force for rapid separation. The high doping of ZrO 2 facilitates effective enrichment of phosphopeptides, even at a very low mass ratio of 1:1000 for tryptic digest of phosphopeptides/non-phosphopeptides, e.g., β-casein/BSA in this particular case. In addition, the PI-Fe x O y -ZrO 2 nanocomposite exhibits better adsorption performance to phosphopeptides with respect to commercial titanium dioxide nanoparticles. The effectiveness of low-abundant phosphopeptides isolation and enrichment from human serum is further identified and demonstrated by means of MALDI-TOF MS and LC-ESI-MS/MS. [ABSTRACT FROM AUTHOR]

Plasmodium mature sexual cycle occurs in the vector mosquitoes and ensures the transmission to a new host. Gametogenesis takes place within minutes in the vector midgut. Gametocytes have to complete a deep nuclear reorganization, quick differentiation, and in the case of male gametocytes, intracytoplasmic flagellum assembly that results in free-swimming microgametes required for macrogamete fertilization. In efforts to improve our knowledge of molecular mechanisms involved in gamete morphogenesis, we carried out a nanoLC/MSMS based quantitative proteomic analysis throughout the xanthurenic acid-induced gametogenesis of the rodent parasite P . berghei . Time-course analyses were performed 7 and 15 min after gametogenesis induction. From 2617 iTRAQ-labelled peptides, 49 proteins were found differentially abundant. Proteins related to RNA translation, DNA, and protein biosynthesis were most prominent and strongly regulated. The energetic metabolic pathway, glycolysis, environmental stress response, RNA/protein biosynthesis, mitosis and axoneme formation, both related to tubulin-associated cytoskeleton dynamic, were predominant regulated cell processes at protein level during the differentiation. Our results also include 26 phosphoproteins in gametocytes/gametes. This first iTRAQ-based proteomic time course analysis of Plasmodium gamete development sheds light on the biological protein orchestration within gametogenesis. Significance Malaria is one of the most serious and widespread parasitic diseases that affected humans in medicine history. The increasing emergence of resistance of parasites from Plasmodium genus to the available antimalarial drugs and the absence of efficient vaccines require an urgent need of development of new therapeutic strategies to fight against that disease. The sexual reproduction is a key step of Plasmodium life cycle and constitutes an attractive target for the development of new therapeutic approaches since it would control malaria based on an inhibition of the parasite transmission to Anopheles , and then to humans. Male and female gamete formation (gametogenesis) is thus a biological event that is determinant for the perpetuation of the parasite in which drastic morphological and metabolic changes occur in a short time interval, resulting in the production of 8 male gametes from a male gametocyte, and fertilization of the female gamete. Development of such transmission-blocking strategies required in deep understanding of the molecular and cellular events associated to gametogenesis. Despite several studies, our understanding on gametogenesis is still incomplete and requires further investigations. This work is the first large-scale quantitative proteomic insight into the P . berghei gamete morphogenesis providing valuable time course data. Plasmodium gametogenesis clearly requires regulation of expression and phosphorylation of proteins belonging to different metabolic pathways and functions, in order to produce mature male and female gametes. [ABSTRACT FROM AUTHOR]

In this study, we designed a gadolinium-based immobilized metal ion affinity chromatography material for the selective enrichment of phosphopeptides. Gadolinium ion was immobilized on the surface of glutathione-coated magnetic nanoparticles through a facile and effective synthetic route. The adsorbent integrated the advantages of superparamagnetism of Fe 3 O 4 core, good biological compatibility of glutathione, and strong interaction between gadolinium ion and phosphopeptides. It was employed to enrich phosphopeptides from standard protein digests coupled with MALDI-TOF MS. Results demonstrated that the adsorbent possessed high selectivity for phosphopeptides, good reusability and reproducibility. Moreover, the material provided selective enrichment of phosphopeptides from real samples including non-fat milk digests and human serum. The developed method exhibited high sensitivity (detection limit of 10 fmol), showing great potential in the detection of low-abundance phosphopeptides in biological samples. [ABSTRACT FROM AUTHOR]

We propose that phosphoproteomic-based studies will radically advance our knowledge about exercise-regulated signaling events. However, these studies use cutting-edge technologies that can be difficult for nonspecialists to understand. Hence, this review is intended to help nonspecialists 1) understand the fundamental technologies behind phosphoproteomic analysis and 2) use various bioinformatic tools that can be used to interrogate phosphoproteomic datasets. [ABSTRACT FROM AUTHOR]

Targeting phosphoinositide 3-kinase (PI3K) has been recognized as an attractive strategy for anticancer therapy. The PI3K is a heterodimer composed of a catalytic subunit p110 and a regulatory subunit p85. Here, instead of targeting the catalytic p110 that has been considered previously, we purposed targeting the peptide-recognition domain SH2 of regulatory p85 with natural medicines obtained by using a peptide scaffold-based screening scheme. In the procedure, a core binding motif was extracted from the cocrystallized complex of a cognate phosphopeptide with the domain, which was considered as basic scaffold to perform high-through virtual screening against a structurally diverse, nonredundant library of natural products. A number of hit compounds with high binding potency to the domain and significant conformational similarity with the peptide scaffold were identified; in vitro affinity assay confirmed that five hits have moderate or high affinity for the domain with measured dissociation constants Kd range between 25 and 360 μM, which are comparable to or even better than that of the cognate phosphopeptide SDpYMNMTP and its core motif peptide pYMNM (Kd = 15 and 32 μM, respectively). Structural analysis and nonbonded comparison of SH2 interactions with phosphopeptides and potent hit compounds revealed that only negatively charged phosphate and, sometime, sulfate can confer domain-binding capability to small-molecule compounds, but carboxylate cannot. A similar binding mode of compounds with phosphopeptide is important for the compounds to have high affinity and specificity. [ABSTRACT FROM AUTHOR]

Phosphopeptides present in a large number of biological regulating mechanisms. It plays a significant role in gene regulation, eukaryotic signal transduction, and metabolic control in a cell. Abnormal phosphorylation can cause various diseases, including cancer. However, phosphoprotein abundances are very low, only 1-2%. In this work, the nanoporous monolith columns were developed for separation and detection of phosphopeptides in phosphoproteome analysis. The nanoporous monolith-based column is prepared inside a silicosteel tubing (100 x 1.02 mm i.d.) by insitu copolymerization reaction of glycidyl methacrylate (GMA) with ethylene dimethacrylate (EDMA). This monolith was further chemically modified by introducing aminomethyl phosphonic acid (AMPA) before immobilization of Ti4+ ion (Ti4+-immobilized). The monolithic column properties, such as morphology, elemental analysis, surface area analysis, permeability and pore distribution were characterized in detail. Such a Ti4+- immobilized nanoporous monolith-based column with immobilization time 3 hours of TiCl4 without glutaraldehyde addition was further applied to separation and detection of phosphopeptides from digested proteins (β-casein and cytochrome-c), and tyrosine phosphorylated peptide samples. The result demonstrated that Ti4+-immobilized nanoporous monolith-based provide higher selectivity and efficiency for selective detection of phosphopeptides using liquid chromatography. [ABSTRACT FROM AUTHOR]

[Display omitted] • Er-Nd-TiO 2 was designed to enrich trace pTyr peptides for pTyr-proteomics. • Er-Nd-TiO 2 possess high sensitivity, high slectivity, less preferences, good reusability. • It can be proposed as an approach to understand the crucial aberrant phosphorylation in AD pathology. The enrichment method of trace phosphotyrosine(pTyr) peptides from complex biological samples is of great concern in pTyr proteomics studies by mass spectrometry. In this study, Er-Nd-TiO 2 (Er3+/Nd3+ co-doped TiO 2) was designed and successfully synthesized by sol–gel method. The Er-Nd-TiO 2 possess high sensitivity (detection limit of 1 × 10−10 M), high selectivity at a low molar ratio of 1:1000:1000 (pTyr peptides: phosphopeptides: non-phosphopeptides), less preferences, and good reusability (8 cycles). The high enrichment efficiency and specificity for phosphotyrosine peptides are ascribed to the synergistic effect of doped Er and Nd, which not only inhibit the growth of TiO 2 nanocrystals inducing a high specific surface area but also have strong affinity and coordination ability toward the phosphate groups of peptides. Moreover, the cation–π interactions between metal cation Er, Nd, Ti and the phenyl ring in an aromatic side chain of tyrosine residue further enhances the specific enrichment of phosphotyrosine peptides by Er-Nd-TiO 2. Compared with commercial TiO 2 , the nanomaterial Er-Nd-TiO 2 had better specific adsorption of p-Tyr peptides and could be applied to the enrichment of p-Tyr peptides in complex samples. [ABSTRACT FROM AUTHOR]

The two non-visual arrestins, arrestin2 and arrestin3, bind hundreds of GPCRs with different phosphorylation patterns, leading to distinct functional outcomes. Structural information on these interactions is available only for very few GPCRs. Here, we have characterized the interactions between the phosphorylated human CC chemokine receptor 5 (CCR5) and arrestin2. We identified several new CCR5 phosphorylation sites necessary for stable arrestin2 complex formation. Structures of arrestin2 in the apo form and complexes with CCR5 C-terminal phosphopeptides, together with NMR, biochemical, and functional assays, revealed three phosphoresidues in a pXpp motif that are essential for arrestin2 binding and activation. The identified motif appears responsible for robust arrestin2 recruitment in many other GPCRs. An analysis of receptor sequences and available structural and functional information provides hints on the molecular basis of arrestin2/arrestin3 isoform specificity. Our findings demonstrate how multi-site phosphorylation controls GPCR⋅arrestin interactions and provide a framework to probe the intricate details of arrestin signaling. [Display omitted] • Phosphorylation sites of the CCR5 C-terminal tail were extensively characterized • The individual sites have distinct effects toward arrestin2 binding and activation • pXpp is a common sequence motif necessary for stable arrestin2 complex formation • GPCR sequence analysis identifies the determinants of arrestin2/arrestin3 specificity Various phosphorylation sites in CCR5 have distinct effects on its interactions with arrestin2. Isaikina and Petrovic et al. identified a pXpp phosphorylation motif in its C-terminal tail, which is crucial for arrestin2 recruitment and common to many other GPCRs. This motif appears as one component of the specific arrestin2 vs. arrestin3 recognition. [ABSTRACT FROM AUTHOR]