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Microtubule-associated protein tau is abnormally phosphorylated and forms the aggregates of paired helical filaments in Alzheimer's disease (AD) and other tauopathies. So far, the relationship and mechanism between the abnormal phosphorylation of tau and fibril formation is still unclear. Therefore, studying the effect of phosphorylation on the structure of tau protein is helpful to elucidate the pathogenic mechanism of tauopathies. It has been shown that pS202/pT205/pS208 triple phosphorylations located in the proline-rich region can promote tau aggregation. In this work, the effect of triple phosphorylations on tau structure was investigated by molecular dynamics simulations combined with multiple analytical methods of trajectories. The results showed that the conformational diversity of G192-T212 fragments decreased after phosphorylation compared with that of the wild-type. Moreover, the dynamic network and hydrogen bond analyses showed that the addition of pS208 phosphorylation can destroy the key hydrogen bonds and the network structure formed centered on pT205 at the C-terminal of the pS202/pT205 double phosphorylated peptide and then destroy the turn structure formed in the region of G207-R211. The destruction of this turn structure is considered to be the main reason for the aggregation of pS202/pT205/pS208 triple phosphorylations. For the pS202/pT205/pS208 triple phosphorylated system, the G207-R211 region is a coil structure, which is more extended and prone to aggregation. In a word, our results reveal the mechanism that pS202/pT205/pS208 triple phosphorylations promote tau aggregation at the atomic level, which can provide useful theoretical guidance for the rational design of effective therapeutic drugs against AD and other tauopathies.

Enhanced conformational sampling, a genetic-algorithm-guided multi-dimensional virtual-system coupled molecular dynamics, can provide equilibrated conformational distributions of a receptor protein and a flexible ligand at room temperature. The distributions provide not only the most stable but also semi-stable complex structures, and propose a ligand–receptor binding process. This method was applied to a system consisting of a receptor protein, 14-3-3ε, and a flexible peptide, phosphorylated Myeloid leukemia factor 1 (pMLF1). The results present comprehensive binding pathways of pMLF1 to 14-3-3ε. We identified four thermodynamically stable clusters of MLF1 on the 14-3-3ε surface, and free-energy barriers among some clusters. The most stable cluster includes two high-density spots connected by a narrow corridor. When pMLF1 passes the corridor, a salt-bridge relay (switching) related to the phosphorylated residue of pMLF1 occurs. Conformations in one high-density spots are similar to the experimentally determined complex structure. Three-dimensional distributions of residues in the intermolecular interface rationally explain the binding-constant changes resultant from alanine–mutation experiment for the residues. We performed a simulation of non-phosphorylated peptide and 14-3-3ε, which demonstrated that the complex structure was unstable, suggesting that phosphorylation of the peptide is crucially important for binding to 14-3-3ε.

Tyrosine phosphorylation (pTyr), much of which occurred on localized multiple sites, initiates cellular signaling, governs cellular functions, and its dysregulation is implicated in many diseases, especially cancers. pTyr-specific sensing is of great significance for understanding disease states and developing targeted anticancer drugs, however, it is very challenging due to the slight difference from serine (pSer) or threonine phosphorylation (pThr). Here we present polyethylenimine-g-phenylguanidine (PEI-PG)-modified nanochannels that can address the challenge. Rich guanidinium groups enabled PEI-PG to form multiple interactions with phosphorylated residues, especially pTyr residue, which triggered the conformational change of PEI-PG. By taking advantage of the "OFF-ON" change of the ion flux arising from the conformational shrinkage of the grafted PEI-PG, the nanochannels could distinguish phosphorylated peptide (PP) from nonmodified peptide, recognize PPs with pSer, pThr, or pTyr residue and PPs with different numbers of identical residues, and importantly could sense pTyr peptides in a biosample. Benefiting from the strong interaction between the guanidinium group and the pTyr side-chain, the specific sensing of pTyr peptide was achieved by performing a simple logic operation based on PEI-PG-modified nanochannels when Ca2+ was introduced as an interferent. The excellent pTyr sensing capacity makes the nanochannels available for real-time monitoring of the pTyr process by c-Abl kinase on a peptide substrate, even under complicated conditions, and the proof-of-concept study of monitoring the kinase activity demonstrates its potential in kinase inhibitor screening.

A highly sensitive electrochemiluminescence (ECL) biosensing method was developed for monitoring casein kinase II (CK2) at subcellular level via bio-bar-code assay. A bio-bar-code probe (h-DNA/AuNPs/p-DNA) prepared by conjugating phosphorylated DNA (p-DNA) and hairpin DNA (h-DNA) onto gold nanoparticles (AuNPs) was used as a carrier for ECL signal reagent (Ru(phen)32+) while a specific peptide was used as a recognition substance. A gold ultramicroelectrode with a diameter of 400 nm was fabricated and then modified with the specific peptide via self-assembly technique to obtain peptide modified gold ultramicroelectrode. The peptide on gold ultramicroelectrode was phosphorylated in the presence of CK2 and adenosine 5′-triphosphate, and then the phosphorylated peptide was integrated with the h-DNA/AuNPs/p-DNA through a process mediated by zirconium cations (Zr4+), and finally Ru(phen)32+ was intercalated into h-DNA. A “signal on” ECL method was developed for the detection of CK2 in the range of 0.005–0.2 U/mL with a detection limit of 0.001 U/mL. Additionally, combined efficient subcellular phosphorylation in vivo with bio-bar-code-based ECL biosensing method, the ECL method was further applied to monitor CK2 at subcellular level without tedious subcellular fractionation. It was found that the concentration of CK2 by inserting the peptide modified gold ultramicroelectrode into the nucleus was higher than that into cytoplasm of HeLa cells. A distinct heterogeneity among CK2 concentrations in single cells was observed for cellular heterogeneity assessment.

CD4+ T-cell activation in infectious diseases and cancer is governed by the recognition of peptides presented on class II human leukocyte antigen (HLA-II) molecules. Therefore, HLA-II ligands represent promising targets for vaccine design and personalized cancer immunotherapy. Much work has been done to identify and predict unmodified peptides presented on HLA-II molecules. However, little is known about the presentation of phosphorylated HLA-II ligands. Here, we analyzed Mass Spectrometry HLA-II peptidomics data and identified 1,113 unique phosphorylated HLA-II ligands. This enabled us to precisely define phosphorylated binding motifs for more than 30 common HLA-II alleles and to explore various molecular properties of phosphorylated peptides. Our data were further used to develop the first predictor of phosphorylated peptide presentation on HLA-II molecules.

The selective and efficient capture of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis. Reported here is a new switchable two-dimensional (2D) supramolecular polymer that can serve as an ideal platform for the enrichment of phos-phopeptides. A well-defined, positively charged metallacycle incorporated into the polymer endows the resultant polymer with a high affinity for phosphopeptides. Importantly, the stimuli-responsive nature of the polymer facilitates switchable binding affinity of phos-phopeptides, thus resulting in excellent performance in phosphopeptide enrichment and separation from model proteins. The polymer has a high enrichment capacity (165 mg/g) and detection sensitivity (2 fmol), high enrichment recovery (88%), excellent specificity, and rapid enrichment and separation properties. Additionally, we have demonstrated the capture of phosphopeptides from the tryptic digest of real bio-samples, thus illustrating the potential of this polymeric material in phosphoproteomic studies.

There is a great desire to relate the patterns of endogenous peptides in blood to human disease and drug response. The best practices for the preparation of blood fluids for analysis are not clear and also relatively few of the peptides in blood have been identified by tandem mass spectrometry. We compared a number of sample preparation methods to extract endogenous peptides including C18 reversed phase, precipitation, and ultrafiltration. We examined the results of these sample preparation methods by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-tandem mass spectrometry (MS/MS) using MALDI-TOF/TOF and electrospray ionization-ion trap. Peptides from solid-phase extraction with C18 in the range of hundreds of femtomoles per spot were detected from the equivalent of 1 μL of serum by MALDI-TOF. We observed endogenous serum peptides from fibrinogen α- and β-chain, complement C3, α-2-HS-glycoprotein, albumin, serine (or cysteine) proteinase inhibitor, factor VIII, plasminogen, immunoglobulin, and other abundant blood proteins. However, we also recorded significant MS/MS spectra from tumor necrosis factor-α-, major histocompatibility complex-, and angiotensin-related peptides, as well as peptides from collagens and other low-abundance proteins. Amino acid substitutions were detected and a phosphorylated peptide was also observed. This is the first time the endogenous peptides of fetal serum have been examined by MS and where peptides from low-abundance proteins, phosphopeptides, and amino acid substitutions were detected.

Phosphorylated peptide from Antarctic krill (P-AKP) was prepared by the dry-heating method with sodium pyrophosphate in order to improve its antioxidant activity and osteogenic activity. P-AKP exhibited more competitive DPPH• and OH• scavenging activities compared to the native Antarctic krill peptide (AKP). In hydrogen peroxide (H2O2)-induced oxidative damage of MC3T3-E1 cells, both AKP and P-AKP pretreatment could dose-dependently improve superoxide dismutase (SOD) and catalase (CAT) activities through attenuating the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) production. Moreover, AKP and P-AKP prevented oxidative stress-induced down regulation of alkaline phosphatase (ALP) activity and matrix mineralization. Particularly, the promoting effects of P-AKP on the enzymatic antioxidant defense system, differentiation and mineralization was higher than that of AKP. These results suggested that phosphorylation might be a promising approach to improve the antioxidant and osteogenic activity of AKP, and P-AKP could be a beneficial agent for attenuating oxidative stress-related bone loss.

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA), was shown to inhibit the kinase activity of IKKα, one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKKα by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKKα and IKKtide, a 20 amino acid peptide substrate derived from IkBα kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKKα and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKKα kinase activity with an IC50 of 0.5 mM, to decrease the Km value from 0.337 mM to 0.402 mM and the Vmax from 0.0257 mM·min−1 to 0.0076 mM·min−1. The computational analyses indicate the region between the KD, ULD and SDD domains of IKKα as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between Km and Ki whereas there is a statistically significant difference between the two Vmax values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide.

Mass spectrometry-based proteomics provides a holistic snapshot of the entire protein set of a living cell on a molecular level. Currently, only a few deep learning approaches that involve peptide fragmentation spectra, which represent partial sequence information of proteins, exist. Commonly, these approaches lack the ability to characterize less studied or even unknown patterns in spectra because of their use of explicit domain knowledge. To elevate unrestricted learning from spectra, we introduce AHLF, a deep learning model that is end-to-end trained on 19.2 million spectra from multiple phosphoproteomic data sets. AHLF is interpretable and we show that peak-level feature importances and pairwise interactions between peaks are in line with corresponding peptide fragments. We demonstrate our approach by detecting post-translational modifications, specifically protein phosphorylation based on only the fragmentation spectrum without a database search. AHLF increases the area under the receiver operating characteristic curve (AUC) by an average of 9.4% on recent phosphoproteomic data compared to the current-state-of-the-art on this task. To show the broad applicability of AHLF we use transfer learning to also detect cross-linked peptides, as used in protein structure analysis, with an AUC of up to 94%. We expect our approach to directly apply to cell signaling and structural biology which use phosphoproteomic and cross-linking data, but in principal any mass spectrometry based study can benefit from an interpretable, end-to-end trained model like AHLF.Availabilityhttps://gitlab.com/dacs-hpi/ahlfContactbernhard.renard@hpi.de

Synthesized peptide substrates have been used for in vitro phosphorylation using purified kinases or cell lysates. For screening assays, a direct readout and comparison among different experimental conditions without using an internal standard would be preferred. In this study, we developed a rapid, quantitative measurement method of multikinase activity based on MALDI-TOF/TOF MS. We combined 8-plex iTRAQ-labeled peptide substrates, solid phase extraction (SPE), and a phosphorylated peptide purification plate to rapidly determine multikinase activity in cell lysates. To enable our platform to be applicable in insulin stimulation and cancer drug inhibition, a list of peptide substrates was designed. By labeling peptide substrates with 8-plex iTRAQ reagents, protein kinase activity in 8 samples could be directly compared using the mass tags on their fragmented ion spectra. The protein amount and incubation time for multikinase activity assays were optimized, and the effect of insulin stimulation and an inhibitory drug on the cellular protein kinase activity was evaluated.

The selective and efficient capture of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis. However, this remains a significant challenge due to the inherently low abundance of these species in complex bio-samples. In this paper, we report a switchable two-dimensional (2D) supramolecular polymer that can serve as an ideal platform for the enrichment of phosphopeptides. A positively charged metallacycle incorporated into the polymer endows the material with a high affinity for phosphopeptides. Importantly, the stimuli-responsive nature of the polymer facilitates switchable binding affinity of phosphopeptides, resulting in its excellent performance in phosphopeptide enrichment and separation from model proteins. The polymer has a high enrichment capacity (165 mg/g) and detection sensitivity (2 fmol), high enrichment recovery (88%), excellent specificity, and rapid enrichment and separation properties. Additionally, we have demonstrated the capture of phosphopeptides from the tryptic digest of real bio-samples illustrating the potential of this polymeric material in phosphoproteomic studies.

Presented herein is a simple, robust, and label-free homogeneous electrochemical sensing platform constructed for the detection of protein kinase activity and inhibition by integration of carboxypeptidase Y (CPY)-assisted peptide cleavage reaction and vertically ordered mesoporous silica films (MSFs). In this sensing platform, the substrate peptide composed of kinase-specific recognized sequence and multiple positively charged arginine (R) residues was ingeniously designed. In the presence of protein kinase, the substrate peptide was phosphorylated and then immediately resisted CPY cleavage. The phosphorylated peptide could be effectively adsorbed on the negatively charged surface of MSFs modified indium-tin oxide (ITO) electrode (MSFs/ITO) by noncovalent electrostatic attraction. The adsorbed peptide was subsequently used as a hamper to prevent the diffusion of electroactive probe (FcMeOH) to the electrode surface through the vertically aligned nanopores, resulting in a detectable reduction of electrochemical signal. As demonstrated for the feasibility and universality of the sensing platform, both protein kinase A (PKA) and casein kinase II (CK2) were selected as the models, and the detection limits were determined to be 0.083 and 0.095 UmL

The effective delivery of bioimaging probes to a selected cancerous tissue has extensive significance for biological studies and clinical investigations. Herein, the peptide functionalized NaGdF4 nanodots (termed as, pPeptide-NaGdF4 nanodots) have been prepared for highly efficient magnetic resonance imaging (MRI) of tumor by formation of Gd-phosphonate coordinate bonds among hydrophobic NaGdF4 nanodots (4.2 nm in diameter) with mixed phosphorylated peptide ligands including a tumor targeting phosphopeptide and a cell penetrating phosphopeptide. The tumor targeting pPeptide-NaGdF4 nanodots have paramagnetic property with ultrasmall hydrodynamic diameter (HD, c.a., 7.3 nm) which greatly improves their MRI contrast ability of tumor and facilitates renal clearance. In detail, the capability of the pPeptide-NaGdF4 nanodots as high efficient contrast agent for in vivo MRI is evaluated successfully through tracking small drug induced orthotopic colorectal tumor (c.a., 195 mm3 in volume) in mouse.

Dephosphorylation of biomolecules under the catalysis of alkaline phosphatase (ALP) is a critical physiological process. Abnormal levels of ALP activity have been associated with a number of diseases; thus, a simple and sensitive assay of ALP activity is highly demanded. Herein, to simulate biological conditions, we labeled a hydrosoluble phosphorylated heptapeptide Gly-Pro-Gly-Asn-p-Tyr-Gly-Ala (pGA) with aminated heptamethine cyanine dye (Cy) to give a low fluorescent labeled peptide Cy-pGA. The synthesized Cy-pGA and Eu3+-doped oxide Y0.6Eu0.4VO4 nanoparticles (NPs) were employed respectively as acceptor and donor to in situ form a non-fluorescent Fluorescence Resonance Energy Transfer (FRET) Cy-pGA-NP system, with the help of the strong interaction between Eu3+ ions in the NPs and phosphate group in Cy-pGA. The breaking of the FRET system of Cy-pGA-NP was triggered by the removal of phosphate group in Cy-pGA catalyzed by ALP and resulting in the release of fluorescent Y0.6Eu0.4VO4 NPs. Thus, the formed Cy-pGA-NP as a sensitive sensor can very well respond to the activity of ALP by measuring the time-resolved fluorescent intensity at near-infrared 617 nm (λ ex = 320 nm, delay time 400 μs). This sensor can not only accurately measure the activity of ALP (1–5 mU/mL) in the designed solutions, but it can also be applied to detect the activity of ALP in biological samples, such as cell lysate and human serum, without the interference of autofluorescent background of biosamples and screen ALP inhibitor by a simple mix-and-measure manner.

A novel photoelectrochemical (PEC) assay is developed for sensitive detection of protein kinase A (PKA) activity based on PKA-catalyzed phosphorylation reaction in solution and signal amplification strategy triggered by PAMAM dendrimer and alkaline phosphatase (ALP). In this strategy, it is noteworthy at this point that PKA phosphorylation was achieved in solution instead of on the surface of the electrode, which has advantages of the good contact in reactants and simple experimental procedure. For immobilizing the phosphorylated peptide (P-peptide) on electrode surface, graphite-like carbon nitride (g-C3N4) and titanium dioxide (TiO2) complex is synthesized and characterized, which plays a significant role for TiO2 conjugating phosphate groups and g-C3N4 providing PEC signal. Subsequently, PAMAM dendrimer and ALP can be captured on P-peptide and TiO2/g-C3N4 modified ITO electrode via interaction between the -COOH groups on the surface of PAMAM dendrimer and the -NH2 groups of peptide and ALP, which can l...

We have developed a novel graphene oxide (GO) sensing platform based on enzymatic phosphorylation for the multiplexed detection of protein kinases. The dye-labeled peptide substrate exhibits strong fluorescence in the absence of kinases. When the biotinylated adenosine 5′-triphosphate co-substrate and kinase are present, the peptide substrate is phosphorylated by kinase, and the phosphorylated peptide product carrying both the biotin site and the dye can be assembled onto streptavidin-coated GO via streptavidin-biotin chemistry, leading to fluorescence quenching. Importantly, since GO is a highly efficient quencher for multiple different fluorophores, the platform allows simultaneous detection of multiple protein kinases using different peptide substrates labeled with corresponding dyes. As a proof-of-concept, we demonstrate that this GO sensing platform can simultaneously and selectively detect protein kinase A, protein kinase Abl and protein kinase Src with low detection limits of 0.005 U/mL, 0.02 U/mL and 0.05 U/mL, respectively. Furthermore, the application of this method for detection of multiple protein kinases in biological samples and protein kinase inhibitor screening has also been demonstrated. This approach holds great promise as a routine tool for the high-throughput screening of protein kinases and their inhibitors.

Antibodies are widely used both in clinical practice and in research. However, the development of methods to increase the ratio of antibodies to recognize phosphorylated proteins remains challenging. In this study, we report a novel and useful method for the efficient production of antibodies for phosphorylated proteins. Based on our previously developed vaccine adjuvant Nap-GDFDFDY, we prepared hydrogels by the Ca2+-induced self-assembly of a phosphorylated peptide gelator Nap-GDFDFpDY. The hydrogel could protect phosphorylated antigens from being dephosphorylated by endogenous phosphatase, thus selectively increasing the ratio of the antibodies for phosphorylated proteins. Our study provides a useful strategy for the production of antibodies to recognize proteins with specific posttranslational modifications.

Phosphorylation is an important post-translational modification on proteins involved in many cellular processes; however, understanding of the regulation and mechanisms of global phosphorylation remains limited. Herein, we utilize self-assembled monolayers on gold for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) with three phosphorylated peptide arrays to profile global phosphatase activity in cell lysates derived from five mammalian cell lines. Our results reveal significant differences in the activities of protein phosphatases on phospho- serine, threonine, and tyrosine substrates and suggest that phosphatases play a much larger role in the regulation of global phosphorylation on proteins than previously understood.

International audience; Spatial localization of biocatalysts, such as enzymes, has recently proven to be an effective process to direct supramolecular self-assemblies in a spatiotemporal way. In this work, silica nanoparticles (NPs) functionalized covalently by alkaline phosphatase (NPs@AP) induce the localized growth of self-assembled peptide nanofibers from NPs by dephosphorylation of Fmoc-FFpY peptides (Fmoc: fluorenylmethyloxycarbonyl; F: phenylalanine; Y: tyrosine; p: phosphate group). The fibrillary nanoarchitecture around NPs@AP underpins a homogeneous hydrogel, which unexpectedly undergoes a macroscopic shape change over time. This macroscopic change is due to a phase separation leading to a dense phase (in NPs and nanofibers) in the center of the vial and surrounded by a dilute one, which still contains NPs and peptide self-assemblies. We thus hypothesize that the phase separation is not a syneresis process. Such a change is only observed when the enzymes are localized on the NPs. The dense phase contracts with time until reaching a constant volume after several days. For a given phosphorylated peptide concentration, the dense phase contracts faster when the NPs@AP concentration is increased. For a given NPs@AP concentration, it condenses faster when the peptide concentration increases. We hypothesize that the appearance of a dense phase is not only due to attractive interactions between NPs@AP but also to the strong interactions of self-assembled peptide nanofibers with the enzymes, covalently fixed on the NPs.

Phosphoprotein-binding domains interact with cognate phosphorylated targets ruling several biological processes. The impairment of such interactions is often associated with disease development, namely cancer. The breast cancer susceptibility gene 1 (BRCA1) C-terminal (BRCT) domain is involved in the control of complex signaling networks of the DNA damage response. The capture and identification of BRCT-binding proteins and peptides may be used for the development of new diagnostic tools for diseases with abnormal phosphorylation profiles. Here we show that designed cyclic β-hairpin structures can be used as peptidomimetics of the BRCT domain, with high selectivity in binding to a target phosphorylated peptide. The amino acid residues and spatial constraints involved in the interaction between a phosphorylated peptide (GK14-P) and the BRCT domain were identified and crafted onto a 14-mer β-hairpin template in silico. Several cyclic peptides models were designed and their binding towards the target peptide and other phosphorylated peptides evaluated through virtual screening. Selected cyclic peptides were then synthesized, purified and characterized. The high affinity and selectivity of the lead cyclic peptide towards the target phosphopeptide was confirmed, and the possibility to capture it using affinity chromatography demonstrated. This work paves the way for the development of cyclic β-hairpin peptidomimetics as a novel class of affinity reagents for the highly selective identification and capture of target molecules.

Selective enrichment techniques are essential for mapping of protein posttranslational modifications (PTMs). Phosphorylation is one of the PTMs which continues to be associated with significant analytical challenges. Particularly problematic are tyrosine-phosphorylated peptides (pY-peptides) resulting from tryptic digestion which commonly escape current chemo- or immuno- affinity enrichments and hence remain undetected. We here report on significant improvements in this regard using pY selective molecularly imprinted polymers (pY-MIPs). The pY-MIP was compared with titanium dioxide (TiO2) affinity based enrichment and immunoprecipitation (IP) with respect to selective enrichment from a mixture of 13 standard peptides at different sample loads. At a low sample load (1pmol of each peptide), IP resulted in enrichment of only a triply phosphorylated peptide whereas TiO2 enriched phosphopeptides irrespective of the amino acid side chain. However, with increased sample complexity, TiO2 failed to enrich the doubly phosphorylated peptides. This contrasted with the pY-MIP showing enrichment of all four tyrosine phosphorylated peptides at 1pmol sample load of each peptide with a few other peptides binding unselectively. At an increased sample complexity consisting of the standard peptides spiked into mouse brain digest, the MIP showed clear enrichment of all four pY- peptides.

This article contains peptides mapping, mass spectrometry and processed data related to the research “Identification and quantification of the phosphorylated ovalbumin by high resolution mass spectrometry under dry-heating treatment” [1]. Fourier transform ion cyclotron mass spectrometry (FTICR MS) was used to investigate the specific phosphorylation sites and the degree of phosphorylation (DSP) at each site. Specifically, phosphorylated peptides were monitored through mass shift on the FTICR MS spectrum. DSP was evaluated through the relative abundance levels of the FTICR MS spectrometry. From these data, the calculation method of DSP was exemplified.

Protein kinases are general and significant regulators in the cell signaling pathway, and it is still greatly desired to achieve simple and quick kinase detection. Herein, we develop a simple and sensitive photoelectrochemical strategy for the detection of protein kinase activity based on the bond between phosphorylated peptide and phosphorylated graphite-like carbon nitride (P-g-C3N4) conjugates triggered by Zr(4+) ion coordination. Under optimal conditions, the increased photocurrent is proportional to the protein kinase A (PKA) concentration ranging from 0.05 to 50 U/mL with a detection limit of 0.077 U/mL. Moreover, this photoelectrochemical assay can be also applied to quantitative analysis of kinase inhibition. The results indicated that the IC50 value (inhibitor concentration producing 50% inhibitor) for ellagic acid was 9.1 μM. Moreover, the developed method is further applied to detect PKA activity in real samples, which contains serum from healthy person and gastric cancer patients and breast tissue from healthy person and breast cancer patients. Therefore, the established protocol provides a new and simple tool for assay of kinase activity and its inhibitors with low cost and high sensitivity.

The Src Homology 2 (SH2) domain is a structurally conserved protein domain that typically binds to a phosphorylated tyrosine in a peptide motif from the target protein. The SH2 domain of C-terminal Src kinase (Csk) contains a single disulfide bond, which is unusual for most SH2 domains. Although the global motion of SH2 domain regulates Csk function, little is known about the relationship between the disulfide bond and binding of the ligand. In this study, we combined X-ray crystallography, solution NMR, and other biophysical methods to reveal the interaction network in Csk. Denaturation studies have shown that disulfide bond contributes significantly to the stability of SH2 domain, and crystal structures of the oxidized and C122S mutant showed minor conformational changes. We further investigated the binding of SH2 domain to a phosphorylated peptide from Csk-binding protein upon reduction and oxidation using both NMR and fluorescence approaches. This work employed NMR, X-ray cryptography, and other biophysical methods to study a disulfide bond in Csk SH2 domain. In addition, this work provides in-depth understanding of the structural dynamics of Csk SH2 domain.

A new diethylenetriamine-derived macrocycle bearing 2-methylpyridyl arms and containing m-xylyl spacers, L, was prepared, and its dinuclear copper(II) and zinc(II) complexes were used as receptors for the recognition in aqueous solution of a phosphorylated peptide derived from a sequence of the STAT3 protein. A detailed study of the acid-base behavior of L and of its complexation properties as well as of the association of the phosphorylated peptide to the receptor was carried out by potentiometry in aqueous solution at 298.2 K and I = 0.10 M in KNO3. The data revealed that the receptor forms stable associations with several protonated forms of the substrate, with constant values ranging from 3.32 to 4.25 log units. The affinity of the receptor for the phosphorylated substrate studied is higher at a pH value where the receptor is mainly in the [Cu2L](4+) form and the pY residue of the substrate is in the dianionic form (pH 6.55). These results, also supported by (31)P NMR studies, showed that the phosphopeptide is bound through the phosphoryl group in a bridging mode. Additionally, the receptor inhibited binding between active (phosphorylated) STAT3 and its target DNA sequence in a dose-dependent manner (IC50 63 ± 3.4 μM) in human nuclear extracts in vitro. Treatment of whole cells with the inhibitor revealed that it is bioactive in living cells and has oncostatic properties that could be interesting for the fight against cancer and other pathologies involving the STAT3 protein.

Microtubule‐associated protein tau becomes abnormally phosphorylated in Alzheimer's disease and other tauopathies and forms aggregates of paired helical filaments (PHF‐tau). AT8 is a PHF‐tau‐specific monoclonal antibody that is a commonly used marker of neuropathology because of its recognition of abnormally phosphorylated tau. Previous reports described the AT8 epitope to include pS202/pT205. Our studies support and extend previous findings by also identifying pS208 as part of the binding epitope. We characterized the phosphoepitope of AT8 through both peptide binding studies and costructures with phosphopeptides. From the cocrystal structure of AT8 Fab with the diphosphorylated (pS202/pT205) peptide, it appeared that an additional phosphorylation at S208 would also be accommodated by AT8. Phosphopeptide binding studies showed that AT8 bound to the triply phosphorylated tau peptide (pS202/pT205/pS208) 30‐fold stronger than to the pS202/pT205 peptide, supporting the role of pS208 in AT8 recognition. We also show that the binding kinetics of the triply phosphorylated peptide pS202/pT205/pS208 was remarkably similar to that of PHF‐tau. The costructure of AT8 Fab with a pS202/pT205/pS208 peptide shows that the interaction interface involves all six CDRs and tau residues 202–209. All three phosphorylation sites are recognized by AT8, with pT205 acting as the anchor. Crystallization of the Fab/peptide complex under acidic conditions shows that CDR‐L2 is prone to unfolding and precludes peptide binding, and may suggest a general instability in the antibody. Proteins 2016; 84:427–434. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

A synthesis of d 5-VX adducted nonapeptide via solid-phase approach has been developed. The d 5-VX peptide could be used as the isotope-labeled internal standard for LC–MS/MS detecting the BuChE-OPNA biomarkers. The Kaiser test was utilized to ensure the right connections of all of the amino acids. This method offers an access to the synthesis and detection of other phosphorylated nonapeptides.

In proteomics, phosphorylation is an important process for protein post-translational modification (PTM), which greatly improves the diversity of proteomes. The PTM regulates almost all physiological and pathological processes such as signal transduction, cell division, proliferation, differentiation and metabolism. The abnormal expression of protein phosphorylation is also associated with cellular metabolic disorders and a range of diseases. However, in mass spectrometry-based phosphorylated peptideomics studies, phosphorylated peptide signals were inhibited by a high abundance of non-phosphorylated peptides; thus, highly selective enrichment was required. In this study, a newly designed material named Fe3O4@MIL(Fe/Ti) was synthesized using a layer-by-layer self-assembly technique that coats the surface of magnetic oxide nanospheres with bimetallic MOF of iron and titanium. The synergistic synthetic coating of the bimetallic MOF gives the material a large surface area and excellent hydrophilicity, which endow the nanoparticles with excellent phosphopeptide enrichment ability, high selectivity (β-casein/BSA molar ratio 1:500), a low detection limit (3 fmol), high recovery rate (85%), strong binding capacity, size exclusion ability, and ideal batch-to-batch repeatability. For comparison, we used Fe3O4@MIL(Fe/Ti) and two single-metal MOF materials Fe3O4@MIL-100(Fe) and Fe3O4@MIL-125(Ti), to enrich α-casein in the middle. Thus, the iron-titanium bimetallic MOF can not only enrich all the phosphorylated peptides enriched by Fe3O4@MIL-100(Fe) and Fe3O4@MIL-125(Ti), but can also specifically enrich four phosphorylated peptides. Encouraged by the excellent results of characterization and standard protein enrichment, we used this material to analyze human serum and found that bimetallic materials can effectively enrich all four phosphorylated peptides and exclude high molecular proteins. These experimental results indicate that the novel bimetallic MOF is a good candidate to analyze protein phosphorylation in complex samples.

Alkaline phosphatase (ALP) is an important biomarker for many diseases. Therefore, the sensitive and accurate detection of ALP activity is essential for fundamental biochemical processes and clinic diagnosis. Herein, we design a fluorescent on–off–on switch for sensitive and visual detection of ALP activity. Meanwhile, mass barcode-modified quantum dots (QDs) amplified the LC-MS/MS detection signal in complex biological samples. Firstly, the QDs were modified with phosphorylated Gly-Gly-Phe-Phe-Tyr (OPO3H2) peptide (GGFFYp) and the mass barcode. The fluorescence of QDs-SS-Yp was quenched by fluorescence resonance energy transfer (FRET) between QDs-SS-Yp and dansyl chloride (DNS). ALP can hydrolyze the phosphorylated peptide to form peptide self-assemblies on the QDs-SS-Yp surfaces. The effective separation distance between the QDs-SS-Yp donor and DNS acceptor becomes larger, restricting FRET between the QDs-SS-Yp and DNS. At this point, the obvious QDs-SS-Yp fluorescence signal can be restored. However, the absence of ALP results in no peptide self-assembly on the QDs-SS-Yp surface and no obvious QDs-SS-Yp fluorescence signal was detected. Therefore, the ALP activity can be analyzed according to the degree of fluorescence restoration by the fluorescence on–off–on switch. Finally, the small tag molecules obtained by cleaving the disulfide bond of the QDs-SS-Yp as a mass barcode were used to amplify the LC-MS/MS detection signal. The proposed approach shows a good linear relationship (from 0.01 to 2.4 U L−1) and has the significant advantage of a low detection limit of 0.001 U L−1.

The human T cell receptor is capable of distinguishing between normal and post-translationally modified peptides. Because aberrant phosphorylation of cellular proteins is a hallmark of malignant transformation, the expression of the phosphorylated epitope could be an ideal antigen to combat cancer without damaging normal tissues. p53 activates transcription factors to suppress tumors by upregulating growth arrest and apoptosis-related genes. In response to DNA damage, p53 is phosphorylated at multiple sites including Ser33 and Ser37. Here, we identified phosphorylated peptide epitopes from p53 that could elicit effective T helper responses. These epitope peptides, p53(22-41/Phospho-S33) and p53(22-41/Phospho-S37), induced T helper responses against tumor cells expressing the phosphorylated p53 protein. Moreover, chemotherapeutic agents augmented the responses of such CD4 T cells via upregulation of phosphorylated p53. The upregulation of phosphorylated p53 expression by chemotherapy was confirmed in in vitro and xenograft models. We evaluated phosphorylated p53 expression in the clinical samples of oropharyngeal squamous cell carcinoma and revealed that 13/24 cases (54%) were positive for phosphorylated p53. Importantly, the lymphocytes specific for the phosphorylated p53 peptide epitopes were observed in the head and neck squamous cell cancer (HNSCC) patients. These results reveal that a combination of phosphorylated p53 peptides and chemotherapy could be a novel immunologic approach to treat HNSCC patients.

In Alzheimer's, the disease-related protein Tau is hyperphosphorylated and aggregates into neurofibrillary tangles (NFT). The cis isomer of the phosphorylated Thr231-Pro232 has been proposed as a precursor of aggregation ('Cistauosis'), but this aggregation scheme is not yet completely accepted. Here, we synthesized peptides comprising a phosphorylated region including Thr231-Pro232 and an aggregation-core region R1 to investigate isomer-specific-aggregation of Tau. The phosphorylated peptide formed amyloid-like aggregation. This aggregation was observed even in the presence of the catalytic domain of the peptidyl-prolyl-isomerase Pin1, which preferentially converts the cis isomer to the trans isomer, but decreased drastically in the presence of the WW domain of Pin1 selectively binding to the trans isomer. These results indicate that the trans isomer is aggregation-prone and that the WW domain of Pin1 effectively inhibits its aggregation.

Background Kawasaki disease (KD) is an acute febrile childhood systemic vasculitis that disturbs coronary arteries. The pathogenesis remains unknown. The study of phosphorylated proteins helps to elucidate the relevant pathophysiological mechanisms of cardiovascular disease. However, few researches explored phosphorylated proteins in KD patients. Methods We compared phosphoprotein profiles of HCAECs stimulated by the serum of KD patients and normal children using iTRAQ technology, TiO2 enrichment phosphorylated peptide and MS analysis. Then we conducted the functional analysis by ClueGO and the biological interaction networking analysis by ReactomeFIViz. Western blotting was performed to identify the hub proteins. Results Our results revealed that phosphorylation of 148 proteins showed different intensities between the two HCAECs groups, which are enriched in MAPK, VEGFR, EGFR, Angiopoietin receptor, mTOR, FAK signaling pathway and so on. Through the Network Analyzer analysis, the hub proteins are CDKN1A, MAPK1 and POLR2A, which were experimentally validated. Conclusion In summary, we provided evidence addressing the valuable phosphorylation signaling that could be useful resource to understand the molecular mechanism and the potential targets for novel therapy of KD. Electronic supplementary material The online version of this article (10.1186/s12872-018-0982-2) contains supplementary material, which is available to authorized users.

β1-adrenergic receptor (Adrb1), a member of the G-protein coupled receptor (GPCR) superfamily, is a critical regulator of heart function. All GPCRs are phosphorylated at multiple sites and the specific pattern of phosphorylation acts as a “barcode” to regulate receptor function and downstream physiological processes in a tissue-specific manner. However, little is known about the location and function of Adrb1 phosphorylation sites in vivo due to the lack of specific antibodies. As a first step to identify the phosphorylation states of Adrb1 and associated functions in the in vivo mouse heart, we developed the following experimental strategy: 1) identification of agonist-dependent Adrb1 phosphorylation sites in isolated perfused mouse heart using advanced phosphoproteomics techniques; 2) definitive assignment of these phosphorylation sites by high-quality mass spectrometry (MS) data obtained from Adrb1- overexpressing HEK 293T cells; 3) generation of knock-in (KI) Mice expressing Adrb1 fused with FLAG-tag at the N-terminus for immunoaffinity purification to reveal phosphorylation status within the living organism; 4) elucidation of phosphorylation levels at specific sites of Adrb1 in KI mouse heart by MS measures of phosphorylated peptide to corresponding unphosphorylated peptide ion intensity ratios. Using this strategy, we identified Ser462 at the C-terminus of Adrb1 as an agonist-dependent phosphorylation site in the perfused mouse heart. We also revealed the basal phosphorylation ratios at Ser274 (0.25), Ser417 (0.55) and Ser462 (0.0023) in KI Mice. These findings provide novel insights into the regulatory mechanisms of Adrb1 function mediated by site-specific phosphorylation.

Aberrant activation of the EGF receptor (EGFR) contributes to many human cancers by activating the Ras-MAPK pathway and other pathways. EGFR signaling is augmented by Src-family kinases, but the mechanism is poorly understood. Here, we show that human EGFR preferentially phosphorylates peptide substrates that are primed by a prior phosphorylation. Using peptides based on the sequence of the adaptor protein Shc1, we show that Src mediates the priming phosphorylation, thus promoting subsequent phosphorylation by EGFR. Importantly, the doubly phosphorylated Shc1 peptide binds more tightly than singly phosphorylated peptide to the Ras activator Grb2; this binding is a key step in activating the Ras-MAPK pathway. Finally, a crystal structure of EGFR in complex with a primed Shc1 peptide reveals the structural basis for EGFR substrate specificity. These results provide a molecular explanation for the integration of Src and EGFR signaling with downstream effectors such as Ras.

Ultra-performance liquid chromatography (UPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) is typically employed for phosphoproteome analysis. Alternatively, capillary zone electrophoresis (CZE) - ESI-MS/MS has great potential for phosphoproteome analysis due to the significantly different migration times of phosphorylated and unphosphorylated forms of peptides. In this work, we systematically compared UPLC-MS/MS and CZE-MS/MS for phosphorylated peptide identifications (IDs) using an enriched phosphoproteome from the MCF-10A cell line. When the sample loading amount of UPLC was 10 times higher than that of CZE (2 μg vs. 200 ng), UPLC generated more phosphorylated peptide IDs than CZE (3,313 vs. 1,783). However, when the same sample loading amounts were used for CZE and UPLC (2–200 ng), CZE-MS/MS consistently and significantly outperformed UPLC-MS/MS in terms of phosphorylated peptide and total peptide IDs. This superior performance is most likely due to the higher peptide intensity generated by CZE-MS/MS. More importantly, compared with UPLC data from 2 μg sample, CZE-MS/MS can identify over 500 unique phosphorylated peptides from 200 ng sample, suggesting that CZE and UPLC are complementary for phosphorylated peptide IDs. With further improved loading capacity via a dynamic pH junction method, 2,313 phosphorylated peptides were identified with single-shot CZE-MS/MS in a 100 min analysis. This number of phosphorylated peptide IDs is over one order of magnitude higher than the number of phosphorylated peptide IDs previously reported by single-shot CZE-MS/MS.

A highly sensitive electrochemiluminescence (ECL) strategy was developed for the protein kinase A (PKA) activity and inhibition assay based on double-quenching of graphene quantum dots (GQDs) ECL by G-quadruplex-hemin DNAzyme and gold nanoparticles (AuNPs). In this strategy, the GQDs were modified onto the indium-tin oxide (ITO) electrode and further assembled with substrate peptide of target protein kinase through covalent coupling, which can exhibit high and stable ECL signal. The AuNPs, functionalized with the phosphorylated DNA and G-quadruplex-hemin DNAzyme via Au-S chemistry, were selected as quenching probes. In the presence of PKA, the peptide on the electrode was phosphorylated and the AuNPs functionalized with the phosphorylated DNA and G-quadruplex-hemin DNAzyme were subsequently integrated onto the phosphorylated peptide by Zr(4+). Owing to the reduction of coreactant H2O2 resulting from G-quadruplex-hemin DNAzyme catalytic reaction and the ECL energy transfer (ECL-RET) between AuNPs and GQDs, the ECL intensity of GQDs was significantly decreased. By taking advantage of the double-quenching effect, this assay can detect PKA with a linear range of 0.05 to 5 U mL(-1) and a detection limit of 0.04 U mL(-1). In addition, the PKA inhibition assay and interferences experiments of CK2 and T4 PNK have been studied respectively. This assay was also successfully applied to PKA assay in serum samples and cell lysates, indicating that the developed method have the potential applications in protein kinase-related biochemical fundamental research and clinical diagnosis.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low density lipoprotein cholesterol (LDL-C) levels. Besides its full-length mature form, multiple variants of PCSK9 have been reported such as forms that are truncated, mutated and/or with posttranslational modifications (PTMs). Previous studies have demonstrated that most of these variants affect PCSK9's function and thereby LDL-C levels. Commercial ELISA kits are available for quantification of PCSK9, but do not allow discrimination between the various forms and PTMs of the protein. To address this issue and given the complexity and wide dynamic range of the plasma proteome, we have developed a mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) for the multiplexed quantification of several forms of circulating PCSK9 in human plasma. Our MSIA-SRM assay quantifies peptides spanning the various protein domains and the S688 phosphorylation site. The assay was applied in two distinct cohorts of obese patients and healthy pregnant women stratified by their circulating LDL-C levels. Seven PCSK9 peptides were monitored in plasma samples: one in the prodomain prior to the autocleavage site at Q152, one in the catalytic domain prior to the furin cleavage site at R218, two in the catalytic domain following R218, one in the cysteine and histidine rich domain (CHRD) and the C-terminal peptide phosphorylated at S688 and unmodified. The latter was not detectable in sufficient amounts to be quantified in human plasma. All peptides were measured with high reproducibility and with LLOQ and LOD below the clinical range. The abundance of 5 of the 6 detectable PCSK9 peptides was higher in obese patients stratified with high circulating LDL-C levels as compared to those with low LDL-C (p < 0.05). The same 5 peptides showed good and statistically significant correlations with LDL-C levels (0.55 < r < 0.65; 0.0002 ⩽ p ⩽ 0.002), but not the S688 phosphorylated peptide. However, this phosphopeptide was significantly correlated with insulin resistance (r = 0.48; p = 0.04). In the pregnant women cohort, none of the peptides were associated to LDL-C levels. However, the 6 detectable PCSK9 peptides, but not PCSK9 measured by ELISA, were significantly correlated with serum triglyceride levels in this cohort. Our results also suggest that PCSK9 circulates with S688 phosphorylated at high stoichiometry. In summary, we have developed and applied a robust and sensitive MSIA-SRM assay for the absolute quantification of all PCSK9 domains and a PTM in human plasma. This assay revealed novel relationships between PCSK9 and metabolic phenotypes, as compared to classical ELISA assays.