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Background Phosphorylation is important in skeletal muscle development, growth, regeneration, and contractile function. Alterations in the skeletal muscle phosphoproteome due to aging have been reported in males; however, studies in females are lacking. We have demonstrated that estrogen deficiency decreases muscle force which correlates with decreased myosin regulatory light chain phosphorylation. Thus, we questioned whether the decline of estrogen in females that occurs with aging might alter the skeletal muscle phosphoproteome. Methods C57BL/6J female mice randomly assigned to a sham-operated (Sham) or ovariectomy (Ovx) group to investigate the effects of estrogen deficiency on skeletal muscle protein phosphorylation in a resting, non-contracting condition. After 16 weeks of estrogen deficiency, the tibialis anterior muscle was dissected and prepped for label-free nano-liquid chromatography tandem mass spectrometry phosphoproteomic analysis. Results We identified 4,780 phosphopeptides in tibialis anterior muscles of ovariectomized (Ovx) and Sham-operated (Sham) control mice. Further analysis revealed 647 differentially regulated phosphopeptides (Benjamini – Hochberg adjusted p-value

Nucleophosmin 1 (NPM1) is a multifunctional protein regulating ribosome biogenesis, centrosome duplication and chromatin remodeling. Being a major nucleolar protein, NPM1 can migrate to the nucleus and the cytoplasm, which is controlled by changes of NPM1 oligomerization and interaction with other cell factors. NPM1 forms a stable pentamer with its N-terminal structured domain, where two nuclear export signals and several phosphorylation sites reside. This domain undergoes dissociation and disordering upon Ser48 phosphorylation in the subunit interface. Recent studies indicated that Ser48 is important for NPM1 interaction with other proteins including 14-3-3, the well-known phosphoserine/phosphothreonine binders, but the structural basis for 14-3-3/NPM1 interaction remained unaddressed. By fusing human 14-3-3ζ with an NPM1 segment surrounding Ser48, which was phosphorylated inside Escherichia coli cells by co-expressed protein kinase A, here we obtained the desired protein/phosphopeptide complex and determined its crystal structure. While biochemical data indicated that the interaction is driven by Ser48 phosphorylation, the crystallographic 14-3-3/phosphopeptide interface reveals an NPM1 conformation distinctly different from that in the NPM1 pentamer. Given the canonical phosphopeptide-binding mode observed in our crystal structure, Ser48 emerges as a conditional binding site whose recognition by 14-3-3 proteins is enabled by NPM1 phosphorylation, disassembly and disordering under physiological circumstances.

The balance between the actions of protein kinases and phosphatases is crucial for neuronal functions, including synaptic plasticity. Although the phosphorylation and dephosphorylation of neuronal proteins are regulated by synaptic plasticity, no systematic analyses of this have yet been conducted. We performed a phosphoproteomic analysis of hippocampal synaptic plasticity using a nano-Acquity/Synapt LC-MS/MS system. Neuronal proteins were extracted from hippocampal tissues and cultured neurons exposed to long-term potentiation (LTP) or long-term depression (LTD). Filter-aided sample preparation (FASP) was performed to remove residual anionic detergents for complete tryptic digestion. Phosphopeptides were then enriched using TiO

Tyrosine phosphorylation (pTyr) regulates various signaling pathways under normal and cancerous states. Due to their low abundance and transient and dynamic natures, systematic profiling of pTyr sites is challenging. Antibody and engineered binding domain-based approaches have been well applied to pTyr peptide enrichment. However, traditional methods have the disadvantage of a long sample preparation process, which makes them unsuitable for processing limited amount of samples, especially in a high-throughput manner. In this study we developed a 96-well microplate-based approach to integrate all the sample preparation steps starting from cell culture to MS-compatible pTyr peptide enrichment in three consecutive 96-well microplates. By assembling an engineered SH2 domain onto a microplate, nonspecific adsorption of phosphopeptides is greatly reduced, which allows us to remove the Ti-IMAC purification and three C18 desalting steps (after digestion, pTyr enrichment, and Ti-IMAC purification) and, therefore, greatly simplifies the entire pTyr peptide enrichment workflow, especially when processing a large number of samples. Starting with 96-well microplate-cultured, pervanadate-stimulated cells, our approach could enrich 21% more pTyr sites than the traditional serial pTyr enrichment approach and showed good sensitivity and reproducibility in the range of 200 ng to 200 μg peptides. Importantly, we applied this approach to profile tyrosine kinase inhibitor-mediated EGFR signaling pathway and could well differentiate the distinct response of different pTyr sites. Collectively, the integrated 96-well microplate-based approach is valuable for profiling pTyr sites from limited biological samples and in a high-throughput manner.

SH2 domains have been recognized as promising targets for various human diseases. However, targeting SH2 domains with phosphopeptides or small-molecule inhibitors derived from bioisosteres of the phosphate group is still challenging. Identifying novel bioisosteres of the phosphate group to achieve favorable

Protein phosphorylation is a prevalent post-translational modification that regulates essentially every aspect of cellular processes. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an extensive offline sample fractionation and a phosphopeptide enrichment method is a best practice for deep phosphoproteome profiling, but balancing throughput and profiling depth remains a practical challenge. We present an online three-dimensional separation method for ultradeep phosphoproteome profiling that combines an online two-dimensional liquid chromatography separation and an additional gas-phase separation. This method identified over 100,000 phosphopeptides (60,000 phosphosites) in HeLa cells during 1.5 days of data acquisition, and the largest HeLa cell phosphoproteome significantly expanded the detectable functional landscape of cellular phosphoproteome.

Normal early erythropoiesis depends on the precise regulation of protein expression and phosphorylation modification. Dysregulation of protein levels or modification contributes to erythroid disorders. To date, the dynamics of protein phosphorylation profiling across human erythroid development is not fully understood. Here, we characterized quantitative proteomic and phosphoproteomic profiling by tandem mass-tagging technology. We systemically built phospho-expression profiling and expression clusters of 11 414 phosphopeptides for human early erythropoiesis. The standardization methods for multitier integrative analyses revealed multiple functional modules of phosphoproteins (e.g., regulation of the G2/M transition) and active phosphorylated signalling (e.g., cell cycle-related pathways). Our further analysis revealed that CDK family members were the main kinases that phosphorylate substrates in erythroid progenitors and identified that CDK9 played an important role in the proliferation of erythroid progenitors. Collectively, our phosphoproteomic profiling, integrative network analysis and functional studies define landscapes of the phosphoproteome and reveal signalling pathways that are involved in human early erythropoiesis. This study will serve as a valuable resource for further investigations of phosphatase and kinase functions in human erythropoiesis and erythroid-related diseases.

BACKGROUND: Within the scope of minimally invasive dentistry, the use of different biocompatible remineralization agents on incisors affected by molar-incisor hypomineralization (MIH) gains importance. OBJECTIVE: To evaluate the effect of casein phosphopeptide amorphous calcium fluoride phosphate (CPP-ACFP) and calcium glycerophosphate (CaGP) in mineral density (MD) of white/creamy and yellow/brown demarcated opacities on incisors affected by MIH by means laser fluorescence (LF). METHODS: As a cross-over, randomized trial, twenty-two children with 167 incisors affected by MIH were recruited and randomly assigned to one of the two different agents and crossed over to other agents with two weeks washout in between. Incisors were examined by using LF at all before and after three months periods. RESULTS: The results of the paired t-tests for determining the period effect between the baseline findings showed significant difference in white/creamy and yellow/brown demarcated opacities of LF values for both groups (p

Human microtubule-associated protein Tau (τ) is abundant in the axons of neurons where it stabilizes microtubule bundles; abnormally hyperphosphorylated τ is a hallmark of Alzheimer's disease (AD) and related tauopathies. The hyperphosphorylation events can be recognized by phosphotyrosine-recognition domain SH2 (Src homology 2) to elicit downstream τ signaling in AD pathology. In this study, a comprehensive binary interaction map (CBIM) of all the 6 τ phosphotyrosine sites with 120 SH2 domains in the human genome was systematically created at structural level using computational analyses and binding assays, from which we were able to identify those of strong and moderate binding pairs of sites to domains. It is found that the SH2-recognition specificity of different τ phosphotyrosine sites has been evolutionally optimized to become roughly orthogonal to each other, and thus these site phosphorylations would regulate different but probably partially overlapped biological functions in τ signaling. Some SH2 groups such as SRC, RIN, PLCG, SOCS and SH2D were revealed to have effective binding potency as compared to others; they could be regarded as potential τ-associated proteins to transduce the downstream signaling. We further determined the systematic binding affinities of 6 τ-phosphopeptides to the 11 SH2 domains in SRC group, from which the FYN-τ

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.\ud \ud

Mutation of an invariant aspartate residue in the binding pocket of 14-3-3ζ isoform to alanine dramatically reduced phosphopeptide binding and induced opening of the binding pocket. Here we use extensive molecular dynamics simulations to understand the role of D124 residue in ligand binding. The simulations show that in the absence of phosphopeptide, the D124A mutation leads to binding pocket reorganization including widening up of the binding pocket at the major groove and repositioning of N173, a key residue that interacts with the main chain of phosphopeptide. These structural changes would interfere with the efficient binding of the peptide, corroborating the experimental observations. Both gain and loss of electrostatic interactions in the form of salt bridges strongly indicate a rearrangement of the network of interactions within the binding pocket. Limited proteolysis coupled mass spectrometry (lip-MS) of the apo and holo forms of wild type (WT) and mutant protein shows a peptide binding helix otherwise buried in the WT protein was particularly accessible to trypsin in the apo form of the mutant protein and the region was mapped to 158-186 amino acid residues of 14-3-3ζ. These results further confirm the dynamic nature of D124A mutant. Unlike other basic residues, the invariant D124 facilitates peptide binding by maintaining the geometry of interacting residues and by enforcing the structural integrity of amphipathic pocket.

A novel hydrophilic porous biocomposite was fabricated by incorporating graphene oxide (GO) @chitosan (CS) foam substrate (GO@CS@ZIF-8 foam) with ZIF-8 crystals in situ via a facile stirring method for simultaneous enrichment of glycopeptides and phosphopeptides from complex biological samples. The experimental results demonstrated that GO@CS@ZIF-8 foam exhibited favorable specificity for simultaneous enrichment of N-glycopeptides and phosphopeptides under the same condition for HRP and β-casein tryptic digest mixtures. The novel material was further applied to enriching both glycopeptides and phosphopeptides simultaneously from 4 μL complex human serum, and 423 N-glycopeptides and 40 phosphopeptides corresponding to 133 glycoproteins and 29 phosphoproteins were identified, respectively.

In this work, we developed an ultra-sensitive CE-MS/MS method for bottom-up proteomics analysis of limited samples, down to sub-nanogram levels of total protein. Analysis of 880 and 88 pg of the HeLa protein digest standard by CE-MS/MS yielded ∼1100 ± 46 and ∼160 ± 59 proteins, respectively, demonstrating higher protein and peptide identifications than the current state-of-the-art CE-MS/MS-based proteomic analyses with similar amounts of sample. To demonstrate potential applications of our ultra-sensitive CE-MS/MS method for the analysis of limited biological samples, we digested 500 and 1000 HeLa cells using a miniaturized in-solution digestion workflow. From 1-, 5-, and 10-cell equivalents injected from the resulted digests, we identified 744 ± 127, 1139 ± 24, and 1271 ± 6 proteins and 3353 ± 719, 5709 ± 513, and 8527 ± 114 peptide groups, respectively. Furthermore, we performed a comparative assessment of CE-MS/MS and two reversed-phased nano-liquid chromatography (RP-nLC-MS/MS) methods (monolithic and packed columns) for the analysis of a ∼10 ng HeLa protein digest standard. Our results demonstrate complementarity in the protein- and especially peptide-level identifications of the evaluated CE-MS- and RP-nLC-MS-based methods. The techniques were further assessed to detect post-translational modifications and highlight the strengths of the CE-MS/MS approach in identifying potentially important and biologically relevant modified peptides. With a migration window of ∼60 min, CE-MS/MS identified ∼2000 ± 53 proteins on average from a single injection of ∼8.8 ng of the HeLa protein digest standard. Additionally, an average of 232 ± 10 phosphopeptides and 377 ± 14 N-terminal acetylated peptides were identified in CE-MS/MS analyses at this sample amount, corresponding to 2- and 1.5-fold more identifications for each respective modification found by nLC-MS/MS methods.

Protein phosphorylation is a crucial regulator of protein and cellular function, yet, despite identifying an enormous number of phosphorylation sites, the role of most is still unclear. Each phosphoform, the particular combination of phosphorylations, of a protein has distinct and diverse biological consequences. Aberrant phosphorylation is implicated in the development of many diseases. To investigate their function, access to defined protein phosphoforms is essential. Materials obtained from cells often are complex mixtures. Recombinant methods can provide access to defined phosphoforms if site-specifically acting kinases are known, but the methods fail to provide homogenous material when several amino acid side chains compete for phosphorylation. Chemical and chemoenzymatic synthesis has provided an invaluable toolbox to enable access to previously unreachable phosphoforms of proteins. In this review, we selected important tools that enable access to homogeneously phosphorylated protein and discuss examples that demonstrate how they can be applied. Firstly, we discuss the synthesis of phosphopeptides and proteins through chemical and enzymatic means and their advantages and limitations. Secondly, we showcase illustrative examples that applied these tools to answer biological questions pertaining to proteins involved in signal transduction, control of transcription, neurodegenerative diseases and aggregation, apoptosis and autophagy, and transmembrane proteins. We discuss the opportunities and challenges in the field.

Highly selective enrichment and sensitive detection of phosphopeptides is pivotal for comprehensive phosphoproteomics analysis; however, it also poses a long-standing challenge. Here, a novel two-dimensional (2D) magnetic bimetallic organic framework (MOF) nanosheet with Zr-O clusters and Ti-O clusters (denoted as the Fe

A multifunctional magnetic cationic MOF composite (Fe3O4@ILI-01@Ti4+) was successfully prepared for the synchronous enrichment of phosphopeptides/glycopeptides.

Despite the fact that bimetallic metal-organic frameworks (MOFs) could afford multiple functionalities by a synergistic effect of individual metallic centers, their intrinsic microporous structure frequently restricts their wide applications with bulky molecules involved. An urgent need is consequently triggered to design bimetallic hierarchical mesoporous MOFs (mesoMOFs). Herein, Zr/Ce mesoMOFs with a uniform pore size of up to 8 nm was successfully synthesized by a copolymer template strategy with the aid of a Hoffmeister ion. The obtained Zr/Ce mesoMOFs feature high porosity, good chemical and thermal stabilities, and tunable element components, and up to 70% Zr could be incorporated into the mesoporous Ce-based framework without deteriorating its crystallinity. Thanks to the synergistic effect of inherent Ce and Zr as well as the large and open pore channels, a broad range of phosphopeptides with different molecule sizes could be effectively checked out, thanks to their simultaneous enrichment and dephosphorylation capabilities. Such an ability to efficiently concentrate phosphopeptides remained intact even in the presence of abundant non-phosphorylated species. The practical detection of phosphopeptides from human serum was also verified, prefiguring the great potentials of bimetallic large-pore mesoMOFs for the proteome applications.

Plant systems science research depends on the dynamic functional maps of the biological substrates of plant phenotypes and host/environment interactions in diverse ecologies. In this context, high-resolution mass spectrometry platforms offer comprehensive insights into the molecular pathways regulated by protein phosphorylation. Reversible protein phosphorylation is a ubiquitous reaction in signal transduction mechanisms in biological systems. In contrast to human and animal biology research, a plethora of experimental options for functional mapping and regulation of plant biology are, however, not currently available. Plant phosphoproteomics is an emerging field of research that aims at addressing this gap in systems science and plant omics, and thus has a large scope to empower fundamental discoveries. To date, large-scale data-intensive identification of phosphorylation events in plants remained technically challenging. In this expert review, we present a critical analysis and overview of phosphoproteomic studies performed in the model plant

Cellular nucleic acid-binding proteins (NABPs), namely, DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in many biological processes. However, extracting NABPs with high efficiency in living cells is challenging, which greatly limited their proteomics analysis and comprehensive characterization. Here, we discovered that titanium (IV) ion-immobilized metal affinity chromatography (Ti4+-IMAC) material could enrich DNA and RNA with high efficiency (96.82 ± 2.67 and 85.75 ± 2.99%, respectively). We therefore developed a Ti4+-IMAC method for the joint extraction of DBPs and RBPs. Through utilizing formaldehyde (FA) cross-linking, DBPs and RBPs were covalently linked to nucleic acids (NAs) and further denatured by organic solvents. After Ti4+-IMAC capture, 2000 proteins were identified in 293T cells, among which 417 DBPs and 999 RBPs were revealed, showing promising selectivity for NABPs. We further applied the Ti4+-IMAC capture method to lung cancer cell lines 95C and 95D, which have different tumor progression abilities. The DNA- and RNA-binding capabilities of many proteins have been dysregulated in 95D. Under our conditions, Ti4+-IMAC can be used as a selective and powerful tool for the comprehensive characterization of both DBPs and RBPs, which might be utilized to study their dynamic interactions with nucleic acids.

Mass spectrometry-based proteomics provide a powerful tool for plant research, allowing global detection of steady-state levels of proteins under a given experimental setup. Here, we provide an optimized protocol for proteomic profiling using tandem mass tag (TMT) labeling followed by liquid chromatography-mass spectrometry (LC-MS/MS) to quantitate phosphopeptides and non-phosphopeptides from the same samples. The outlined protocol comprises a series of successive steps, namely, SDS (sodium dodecyl sulfate) protein extraction, protein precipitation, digestion, TMT labeling, phosphopeptide enrichment, high pH reversed-phase fractionation, LC-MS/MS analysis, protein identification, and data analysis. Our proteome-scale protocol requires 0.1 mg protein per sample and allows for the reliable and accurate quantification of more than 8000 proteins in Arabidopsis plant samples across multiple conditions, including low abundant peptides.

Protein phosphorylation is one of the most widely studied posttranslational modifications, and its role in signal transduction has gained particular attention. The relatively low abundance of the phosphorylated form of proteins makes identification by mass spectrometry challenging in the absence of selective enrichment. Titanium oxide-based enrichment of phosphopeptides in the presence of acidic modifiers is highly selective and makes it technically possible to detect thousands of phosphopeptides in a single sample by liquid chromatography-mass spectrometry. In this chapter, we describe a detailed protocol for the selective enrichment of microsomal and cytosolic phosphopeptides from Arabidopsis seedlings. The resulting phosphopeptide fractions enable routine identification of several thousands of phosphopeptide spectra per sample by mass spectrometry.

In this chapter, detailed procedures for stable isotope labeling with amino acids in cell culture, SILAC labeling of yeast auxotroph, optimization and evaluation of phosphopeptide enrichment, and sample preparation and analysis by high-resolution LC-MS/M, identification of phosphosites, and quantification methods are described.We report methods for the application of double SILAC to yeast using a combination of labeled lysine and labeled arginine.The combination of SILAC-based quantitation with phosphopeptides enrichment by TiOsub2/subin a batch that enables measurement of protein posttranslational modifications is a powerful application to analyze the global phosphoproteome for studies in signaling pathways.

INTRODUCTION. Protein phosphorylation is a primary mechanism of signal transduction in cellular systems. Isobaric tagging can be used to investigate alterations in phosphorylation events in sample multiplexing experiments where quantification extends across all conditions. As such, innovations in tandem mass tag methods can facilitate the expansion of the depth and breadth of phosphoproteomics analyses. AREAS COVERED. This review discusses the current state of tandem mass tag-centric phosphoproteomics and highlights advances in reagent chemistry, instrumentation, data acquisition, and data analysis. We stress that approaches for phosphoproteomic investigations require high-specificity enrichment, sensitive detection, and accurate phosphorylation site localization. EXPERT OPINION. Tandem mass tag-centric phosphoproteomics will continue to be an important conduit for our understanding of signal transduction in living organisms. We anticipate that progress in phosphopeptide enrichment methodologies, enhancements in instrumentation and data acquisition technologies, and further refinements in analytical strategies will be key to the discovery of biologically relevant data from phosphoproteomics studies.

Protein phosphorylation in prokaryotes has gained more attention in recent years as several studies linked it to regulatory and signaling functions, indicating importance similar to protein phosphorylation in eukaryotes. Studies on bacterial phosphorylation have so far been conducted using manual or HPLC-supported phosphopeptide enrichment, whereas automation of phosphopeptide enrichment has been established in eukaryotes, allowing for high-throughput sampling. To facilitate the prospect of studying bacterial phosphorylation on a systems level, we here established an automated Ser/Thr/Tyr phosphopeptide enrichment workflow on the Agilent AssayMap platform. We present optimized buffer conditions for TiO2 and Fe(III)-NTA-IMAC cartridge-based enrichment and the most advantageous, species-specific loading amounts for Streptococcus pyogenes, Listeria monocytogenes, and Bacillus subtilis. For higher sample amounts (≥250 μg), we observed superior performance of the Fe(III)-NTA cartridges, whereas for lower sample amounts (≤100 μg), TiO2-based enrichment is equally efficient. Both cartridges largely enriched the same set of phosphopeptides, suggesting no improvement of peptide yield by the complementary use of the two cartridges. Our data represent, to the best of our knowledge, the largest phosphoproteome identified in a single study for each of these bacteria.

Elevated immunity to cancer-expressed antigens can be detected in people with no history of cancer and may contribute to cancer prevention. We have previously reported that MHC-restricted phosphopeptides are cancer-expressed antigens and targets of immune recognition. However, the extent to which this immunity reflects prior or ongoing phosphopeptide exposures was not investigated. In this study, we found that preexisting immune memory to cancer-expressed phosphopeptides was evident in most healthy donors, but the breadth among donors was highly variable. Although three phosphopeptides were recognized by most donors, suggesting exposures to common microbial/infectious agents, most of the 205 tested phosphopeptides were not recognized by peripheral blood mononuclear cells (PBMC) from any donor and the remainder were recognized by only 1 to 3 donors. In longitudinal analyses of 2 donors, effector immune response profiles suggested active reexposures to a subset of phosphopeptides. These findings suggest that the immunogens generating most phosphopeptide-specific immune memory are rare infectious agents or incipient cancer cells with distinct phosphoproteome dysregulations, and that repetitive immunogenic exposures occur in individual donors. Phosphopeptide-specific immunity in PBMCs and tumor-infiltrating lymphocytes from ovarian cancer patients was limited, regardless of whether the phosphopeptide was expressed on the tumor. However, 4 of 10 patients responded to 1 to 2 immunodominant phosphopeptides, and 1 showed an elevated effector response to a tumor-expressed phosphopeptide. As the tumors from these patients displayed many phosphopeptides, these data are consistent with lack of prior exposure or impaired ability to respond to some phosphopeptides and suggest that enhancing phosphopeptide-specific T-cell responses could be a useful approach to improve tumor immunotherapy.

The systematic and in-depth study of phosphoproteome rely on highly reproducible and specific phosphopeptide enrichment methods. At present, a variety of enrichment methods have been developed based on different principles, and these methods often display different selectivity and specificity. It is therefore very important to select the most suitable enrichment method according to different research purposes. This review summarized the phosphopeptide enrichment based on affinity chromatography, immunoprecipitation, chemical derivatization, chromatography and other newly developed methods. The advantages and disadvantages of these methods, as well as the related optimization and improvement strategies, were discussed in detail. In addition, we also briefly summarized the progress of the combination of phosphopeptide enrichment and fractionation methods developed in recent years.

Tuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis (Mtb) infections drives an urgent need to exploit new drug targets. One such target is the ATP-dependent protease ClpC1P1P2, which is strictly essential for viability. However, few proteolytic substrates of mycobacterial ClpC1P1P2 have been identified to date. Recent studies in Bacillus subtilis have shown that the orthologous ClpCP protease recognizes proteolytic substrates bearing post-translational arginine phosphorylation. Several lines of evidence suggest that ClpC1P1P2 similarly recognizes phosphoarginine-bearing proteins, but the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of post-translational phosphoarginine modifications in Mycolicibacterium smegmatis (Msm), a nonpathogenic surrogate of Mtb. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identify arginine phosphosites on a diverse collection of targets within the Msm proteome. Physicochemical and functional characterization of targets suggest that arginine phosphorylation is applied in a sequence-independent manner as part of a proteome-wide quality control pathway. Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/438432v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@607043org.highwire.dtl.DTLVardef@16a4614org.highwire.dtl.DTLVardef@1efaf5org.highwire.dtl.DTLVardef@1ee2247_HPS_FORMAT_FIGEXP M_FIG C_FIG

Among all post-translational modifications of proteins, phosphorylation is one of the most common and most studied. Since plants are sessile organisms, many physiological processes on which their survival depends are regulated by phosphorylation and dephosphorylation. Understanding the extent to which a plant proteome is phosphorylated at specific developmental stages and/or under certain environmental conditions is essential for identifying molecular switches that regulate physiological processes and responses. While most phosphoproteomic workflows proposed in the literature provide tools to exclusively analyze phosphorylated proteins, it is imperative to examine both the proteome and the phosphoproteome to reveal the true complexity of a biological process. Here we describe a mass spectrometry-based phosphoproteomics workflow to analyze both total and phosphorylated proteins. Our method includes phenol-based protein extraction, as well as techniques to measure the quantity and quality of protein extracts. In addition, we compare in detail the efficiency and suitability of in-gel and in-solution trypsin digestion methods. A metal oxide affinity chromatography technique for rapid and efficient enrichment of phosphorylated peptides and an LC-MS/MS method for analysis of the phosphorylated peptides are described. Finally, we present and discuss the results generated by applying this workflow to our study of the C

To investigate the effects of bleaching with 35% hydrogen peroxide on the structure of tooth enamel and the role of two remineralizing agents for their potential to remineralize any damaged regions of enamel.Freshly extracted 32 mature permanent central incisors were selected and sectioned at the level of the cemento-enamel junction. The teeth were divided into four groups consisting of eight teeth each: No bleaching (control) [Group 1], bleaching with 35% hydrogen peroxide [Group 2], bleaching with 35% hydrogen peroxide followed by application of casein phosphopeptide amorphous calcium phosphate fluoride paste [Group 3], and bleaching with 35% hydrogen peroxide followed by application of xylitol-coated calcium phosphate fluoride varnish [Group 4]. The enamel surfaces were analyzed under the scanning electron microscope and quantitative energy dispersive X-ray analysis.Results were statistically analyzed by one-way analysis of variance and Tukey's posthoc test. Group 2 revealed changes in enamel surface morphology and a statistically significant decrease in mineral content. Groups 3 and 4 showed statistically significant remineralization potential. Intergroup comparison showed that samples in Group 4 had a higher mineral content compared to Group 3.The application of the tested remineralizing agents following bleaching was effective in repairing the enamel surface morphology with higher efficacy for the fluoride varnish product. Since bleaching regimes with high concentrations of hydrogen peroxide adversely affect the enamel surface, these findings can translate to clinical practice to reduce the long-term damaging effects of tooth bleaching.

Phosphorylation and glycosylation of proteins, two of the most widely studied post-translational modifications (PTMs), have shown increasing potential in the early non-invasive diagnosis, prognosis, and therapeutic evaluation of diseases. Besides regulating the function of cell membranes and intracellular signal transduction, protein phosphorylation participates in mitochondrial function and cellular and transcriptional metabolism. Protein glycosylation plays an important role in both intracellular and extracellular signal transduction and intracellular endocytosis. Aberrant phosphorylation and glycosylation of proteins are frequently observed in clinical proteomic studies and in the discovery of disease-related biomarkers. There are generally three methods for detecting protein phosphorylation/glycosylation: isotope radiolabeling, western blotting, and mass spectrometry. Mass spectrometry has become the most important and advantageous detection method due to its high throughput and time- and labor-efficiency. However, phosphopeptides and glycopeptides have low stoichiometry and ionization efficiency, and a large number of non-phosphopeptides and -glycopeptides interference. These issues make it difficult to directly detect phosphopeptides and glycopeptides by mass spectrometry. Therefore, the enrichment of phosphopeptides and glycopeptides before mass spectrometry detection is a key step. At present, a variety of materials have been developed for enrichment studies of phosphopeptides and glycopeptides. For example, immobilized metal affinity (IMAC) and metal oxide affinity chromatography (MOAC) methods are mostly used for the enrichment of phosphopeptides. The IMAC mainly uses positively charged metal ions and negatively charged phosphate groups to attract each other for the purpose of enriching phosphopeptides. MOAC materials rely on the chelation of metal atoms and phosphate oxygens to capture phosphopeptides. IMAC and MOAC materials rely on strong interactions between metals and phosphate groups, which often lead to difficult elution. The enrichment method for glycopeptides is mainly based on the difference in hydrophilicity between glycopeptides and non-glycopeptides, which are mainly enriched by hydrophilic interaction chromatography (HILIC). In addition, materials containing compounds such as boronic acid and lectin materials are also widely used for the separation and enrichment of glycopeptides. Smart responsive materials have also been successively reported for the enrichment of phosphopeptides and glycopeptides due to their unique responsiveness and reversibility. Smart responsive materials can respond to external stimuli; undergo structural and property changes; and convert signals such as optical, electrical, thermal, and mechanical into biochemical signals. Responsive molecules are a prerequisite for determining the response properties of smart responsive materials, and their reversible isomerization under different stimuli (such as temperature, pH, light, mechanical stress, and electromagnetic field) will lead to dynamic changes in the physical and chemical properties of materials. Compared with traditional materials, smart responsive materials can be reversibly "turned on" and "off" with better controllability. Exogenous stimuli, including temperature, light, ultrasound, electromagnetic field, and mechanical stress, can be implemented in a specific time and space. Exogenous responsive materials do not depend on changes in the reaction system itself and are non-invasive. Enzymes, pH, redox, solution polarity, and ionic strength are endogenous stimuli. Endogenous responsive materials depend on changes in the reaction system itself, and sometimes the regulation process requires the introduction of other chemicals into the reaction system. The identification, capture, and release of phosphopeptides or glycopeptides can be achieved by modulating the interactions between smart responsive materials and phosphopeptides or glycopeptides (such as hydrogen bonds, and electrostatic and hydrophobic interactions). This review classifies smart responsive materials according to the types of stimuli, which are specifically divided into exogenous and endogenous responsive materials. The enrichment of phosphopeptides and glycopeptides of exogenous/endogenous responsive materials and endogenous/exogenous co-responsive materials are summarized. In addition, we discuss the development prospects of smart responsive materials in the enrichment of phosphopeptides and glycopeptides, and also raised the challenges existing in the application of smart responsive materials in other protein post-translational modifications.

Gout is a painful form of inflammatory arthritis characterized by the deposition of monosodium urate (MSU) crystals in the joints. The aim of this study was to investigate the effect of peptide P140 on the inflammatory responses in crystal-induced mouse models of gout and cell models including MSU-treated human cells. Injection of MSU crystals into the knee joint of mice induced neutrophil influx and inflammatory hypernociception. Injection of MSU crystals subcutaneously into the hind paw induced edema and increased pro-inflammatory cytokines levels. Treatment with P140 effectively reduced hypernociception, the neutrophil influx, and pro-inflammatory cytokine levels in these experimental models. Furthermore, P140 modulated neutrophils chemotaxis in vitro and increased apoptosis pathways through augmented caspase 3 activity and reduced NFκB phosphorylation. Moreover, P140 increased the production of the pro-resolving mediator annexin A1 and decreased the expression of the autophagy-related ATG5-ATG12 complex and HSPA8 chaperone protein. Overall, these findings suggest that P140 exerts a significant beneficial effect in a neutrophilic inflammation observed in the model of gout that can be of special interest in the design of new therapeutic strategies. ispartof: CELLS vol:11 issue:23 ispartof: location:Switzerland status: published

Herein, we show intranuclear nanoribbons formed upon dephosphorylation of leucine-rich L- or D-phosphopeptide catalyzed by alkaline phosphatase (ALP) to selectively kill osteosarcoma cells. Being dephosphorylated by ALP, the peptides are first transformed into micelles and then converted into nanoribbons. The peptides/assemblies first aggregate on cell membranes, then enter cells via endocytosis, and finally accumulate in nuclei (mainly in nucleoli). Proteomics analysis suggests that the assemblies interact with histone proteins. The peptides kill osteosarcoma cells rapidly and are nontoxic to normal cells. Moreover, the repeated stimulation of the osteosarcoma cells by the peptides sensitizes the cancer cells rather than inducing resistance. This work not only illustrates a novel mechanism for nucleus targeting, but may also pave a new way for selectively killing osteosarcoma cells and minimizing drug resistance.

Dysregulation of protein phosphatases is associated with the progression of various human diseases and cancers. Herein, a photoelectrochemical (PEC)-quartz crystal microbalance (QCM) dual-mode sensing platform was developed for protein tyrosine phosphatase 1B (PTP1B) activity assay based on bifunctional magnetic Fe

In this study, new graphene-based IMAC nanocomposites for phosphopeptide enrichment were prepared according to the guideline of our new design strategy. Superhydrophilic polyethyleneimine (PEI) was introduced, to which a phosphonate-functionalized ionic liquid (PFIL) was covalently bound, to form superhydrophilic and cationic surface layers with high densities of nitrogen atoms, phosphonate functional groups, and high-loading metal ions. Due to the combined features of superhydrophilicity, flexibility, highly dense metal binding sites, large surface area and excellent size-exclusion effect, the fabricated nanocomposite G@mSiO

Non-functioning pituitary tumours (NF-PitNETs) are common intracranial benign neoplasms that can exhibit aggressive behaviour by invading neighbouring structures and, in some cases, have multiple recurrences. Despite resulting in severe co-morbidities, no predictive biomarkers of recurrence have been identified for NF-PitNETs. In this study we have used high-throughput mass spectrometry-based analysis to examine the phosphorylation pattern of different subsets of NF-PitNETs. Based on histopathological, radiological, surgical and clinical features, we have grouped NF-PitNETs into non-invasive, invasive, and recurrent disease groups. Tumour recurrence was determined based on regular clinical and radiological data of patients for a mean follow-up of 10 years (SD ± 5.4 years). Phosphoproteomic analyses identified a unique phosphopeptide enrichment pattern which correlates with disease recurrence. Candidate phosphorylated proteins were validated in a large cohort of NF-PitNET patients by western blot and immunohistochemistry. We identified a cluster of 22 phosphopeptides upregulated in recurrent NF-PitNETs compared to non-invasive and invasive subgroups. We reveal significant phosphorylation of the β-catenin at Ser552 in recurrent and invasive NF-PitNETs, compared to non-invasive/non-recurrent NF-PitNET subgroup. Moreover, β-catenin pSer552 correlates with the recurrence free survival among 200 patients with NF-PitNET. Together, our results suggest that the phosphorylation status of β-catenin at Ser552 could act as potential biomarker of tumour recurrence in NF-PitNETs.

Efficient phosphoproteomic analysis of small amounts of biological samples (e.g. tissue biopsies) requires carefully selected enrichment and purification steps prior to the nanoflow HPLC-MS/MS analysis. Solid-phase extraction (SPE) is one of the most commonly used approaches for sample preparation. Several stationary phases are available for peptide SPE purification, however, most of the published methods are not optimized to provide good recoveries of phosphorylated peptides. Our goal was to investigate the performance of 13 self-packed and 3 commercial centrifugal SPE cartridges/spin tips, thus enhancing the efficiency of the phosphoproteomic analysis of small amounts of complex protein mixtures. Eight reversed-phase (RP), five graphite, two ion-exchange, and one hydrophilic-lipophilic balance (HLB) stationary phase were evaluated. Two RP, one graphite, and the HLB self-packed centrifugal SPE tips provided excellent results for the purification of 1 µg tissue and cell line digests. Using these methods, the sample loss was significantly reduced compared to one of the commercial SPE methods, 22-58% more unique phosphopeptides were identified, and the recovery was higher by 132-155%.

Many pathological conditions are caused by dysregulation of cell signaling, which can generate a cascade of abnormal responses and completely change the functions of a cell or tissue. A large portion of the regulation of these signals is via protein phosphorylation, in which cell responses can be activated or inhibited. Proteins that are both downstream and upstream of a phosphorylated protein can be modified, altering metabolism and other biological processes. Recently, the number of phosphoproteomic studies based on mass spectrometry has increased, constantly aiming to obtain a higher coverage of proteins and increase the number and location of their phospho-sites, as well as better understand their respective phosphorylation states. In this way, it is possible to better understand biological processes as a whole and their roles in cellular dysfunctions and diseases. To study changes at the phosphoproteome level, the stochiometric imbalance between the non-phosphorylated and phosphorylated peptides must be overcome, since higher quantities and comparatively better ionization of non-phosphorylated peptides can suppress the ion signals of the phosphorylated peptides. It is for this reason that phosphophopeptides are rarely found in samples that did not pass through a phospho-enrichment step, highlighting the importance of performing enrichment steps in phosphoproteomic studies. The numbers of identified phosphopeptides and phosphorylation sites are extremely important to the quality of a phosphoproteomic analysis; therefore, the efficiency of the enrichment process is critical. Here, phospho-enrichment techniques are presented to offer insight into the applicability of these methods to different experiment types and consequently support the growth of phosphoproteomic studies overall.

The flexible and controlled synthesis of metal-organic framework (MOF)-derived hybrid nanostructures is of great significance in fine tuning of their enrichment performance in large-scale and in-depth phosphoproteome analysis. Herein, a magnetic guanidyl-functionalized MOF hybrid coating with multiaffinity sites, denoted as Fe

A guanidine-functionalized (GF) covalent organic framework (COF) nanocomposite has been developed by a post-synthetic approach for specific capture and separation of phosphopeptides and exosomes. The abundant binding sites on COF can immobilize a large number of gold nanoparticles (AuNPs), which can be used to react with amino groups to graft polyethyleneimine (PEI). Finally, Fe

3-O-trans-caffeoyloleanolic acid (COA) is a pentacyclic triterpenoid compound, with significant anti-inflammatory effects. In this study, we report the protective effects of COA on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and explored its mechanism of action. LPS was used to construct in vivo mouse ALI models to observe the effects of COA pretreatment on lung pathology, inflammation, and oxidative stress. In vitro, mouse alveolar macrophages MH-S cells were cultured and stimulated with LPS to investigate the effects of COA pretreatment on inflammation and oxidative stress. Western blotting was used to investigate the expression of iNOS, TLR4, p-p65, p-AKT, and p-PI3K from in vivo and in vitro samples. The results showed that COA significantly improved lung injury, inhibited neutrophil infiltration, prevented macrophage infiltration, inhibited the release of inflammatory factors, reduced oxidative stress, and down-regulated the expression of iNOS, TLR4, p-p65, p-AKT, and p-PI3K in ALI mice caused by LPS. In vitro, COA inhibited the release of inflammatory factors, reduced oxidative stress, and down-regulated the expression of iNOS, TLR4, p-p65, p-AKT, and p-PI3K in MH-S cells stimulated with LPS. Of interest, the protective effects of COA were significantly attenuated in MH-S cells pretreated with the PI3K phosphopeptide activator 740Y-P with no effect on TLR4 expression observed. Taken together, these findings confirm the protective effects of COA on ALI. We further demonstrate that the anti-inflammation and antioxidant effects of COA are mediated through its effects on PI3K/AKT and potentially TLR4.

Mass spectrometry-based profiling of the phosphoproteome is a powerful method of identifying phosphorylation events at a systems level. Most phosphoproteomics studies have used data-dependent acquisition (DDA) mass spectrometry as their method of choice. In this Perspective, we review some recent studies benchmarking DDA and DIA methods for phosphoproteomics and discuss data analysis options for DIA phosphoproteomics. In order to evaluate the impact of data-dependent and data-independent acquisition (DIA) on identification and quantification, we analyze a previously published phosphopeptide-enriched data set consisting of 10 replicates acquired by DDA and DIA each. We find that though more unique identifications are made in DDA data, phosphopeptides are identified more consistently across replicates in DIA. We further discuss the challenges of identifying chromatographically coeluting phosphopeptide isomers and investigate the impact on reproducibility of identifying high-confidence site-localized phosphopeptides in replicates.

Enrichment of phosphopeptides before mass spectrometry (MS) analysis is essential due to the limitations of low abundance and poor ionization efficiency in complex biological samples. Immobilized metal affinity chromatography (IMAC), especially titanium ion (Ti

Phosphorylation is one of the most important protein post-translational modifications, which possesses dramatic regulatory effects on the function of proteins. In consideration of the low abundance and low stoichiometry of phosphorylation and non-specific signal suppression, efficient capture of the phosphoproteins from complex biological samples is critical to meet the need for protein profiling. In this work, a facile preparation of titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles was developed for the enrichment of intact phosphoproteins. The prepared magnetic nanoparticles were characterized by various instruments and had a spherical shape with an average diameter of 300 nm. The adsorption isotherms were investigated and the maximum capacity for β-casein was calculated to be 961.5 mg/g. Standard protein mixtures and biological samples (non-fat milk and human serum) were selected to test the enrichment performance. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated the excellent enrichment performance with high selectivity. With the superparamagnetic property, titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles were convenient for the practical application and clinical promotion, thus having a promising prospect in the field of phosphoprotein research.

A detailed understanding of the sequence preference surrounding phosphorylation sites is essential for deciphering the function of the human phosphoproteome . Whereas the mechanisms for substrate site recognition by kinases are relatively well understood, the selection mechanisms for the corresponding phosphatases pose several obstacles. However, multiple pieces of evidence point towards a role of the amino acid sequence in the direct vicinity of the phosphorylation site for recognition by phosphatase enzymes. Peptide library-based studies for enzymes attaching posttranslational modifications (PTMs) are relatively straight forward to carry out. However, studying enzymes removing PTMs pose a challenge in that libraries with a PTM attached are needed as a starting point. Here, we present our methodology using large synthetic phosphopeptide libraries to study the preferred sequence context of protein phosphatases. The approach, termed "phosphopeptide library dephosphorylation followed by mass spectrometry" (PLDMS), allows for the exact control of phosphorylation site incorporation and the synthetic route is capable of covering several thousand peptides in a single tube reaction. Furthermore, it enables the user to analyze MS data tailored to the needs of a specific library and thereby increase data quality. We therefore expect a wide applicability of this technique for a range of enzymes catalyzing the removal of PTMs.

A newly designed TEMPO-FRIPS reagent, 4-(2,2,6,6-tetramethylpiperidine-1-oxyl) methyl benzyl succinic acid N-hydroxysuccinimide ester or p-TEMPO–Bn–Sc–NHS, was synthesized to achieve single-step free radical-initiated peptide sequencing mass spectrometry (FRIPS MS) for a number of model peptides, including phosphopeptides. The p-TEMPO–Bn–Sc–NHS reagent was conjugated to target peptides, and the resulting peptides were subjected to collisional activation. The peptide backbone dissociation behaviors of the MS/MS and MS3 experiments were monitored in positive ion mode. Fragment ions were observed even at the single-step thermal activation of the p-TEMPO–Bn–Sc–peptides, showing mainly a-/x- and c-/z-type fragments and neutral loss ions. This confirms that radical-driven peptide backbone dissociations occurred with the p-TEMPO–Bn–Sc–peptides. Compared to the previous version of the TEMPO reagent, i.e., o-TEMPO–Bz–C(O)–NHS, the newly designed p-TEMPO–Bn–Sc–NHS has better conjugation efficiency for the target peptides owing to its improved structural flexibility and solubility in the experimental reagents. An energetic interpretation using the survival fraction as a function of applied normalized collision energy (NCE) ascertained the difference in the thermal activation between p-TEMPO–Bn–Sc– and o-TEMPO–Bz–C(O)– radical initiators. This study clearly demonstrates that the application of the p-TEMPO–Bn–Sc– radical initiator can improve the duty cycle, and this FRIPS MS approach has the potential to be implemented in proteomics studies, including phosphoproteomics.

This study aimed to investigate the efficacy of three remineralizing agents on dental erosion in primary teeth.Forty primary molars were randomly divided into four groups (n = 10 each): self-assembling peptide (Psub11-4/sub), casein phosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP), sodium fluoride (NaF) and artificial saliva (AS; control). The erosion-like formation was created by immersing the samples in citric acid (4 × 2 min, pH 2.3) and AS (4 × 2 h, pH 7). The eroded samples were then treated with remineralizing agents and subjected to further erosion consisting of 15 cycles (3x/8-h interval) of immersion in citric acid and AS for 6 s each. Alterations in the mineral content and morphology of the samples were quantified using a microhardness tester and atomic force microscope.All agents had a significant remineralization effect on eroded primary tooth enamel. After further erosive challenge, enamel loss in the CPP-ACFP group was found to be significantly lower than in all other groups, and no significant difference was found between the Psub11-4/suband NaF groups.This study showed that all tested materials had remineralization ability, and CPP-ACFP had a superior effect in inhibiting enamel loss due to dental erosion in primary teeth. © 2022 Australian Dental Association.

Protein phosphorylation is an essential post-translational modification that regulates multiple cellular processes. Due to their low stoichiometry and ionization efficiency, it is critical to efficiently enrich phosphopeptides for phosphoproteomics. Several phosphopeptide enrichment methods have been reported; however, few studies have comprehensively compared different TiO

Microcystin-LR (MC-LR) is a kind of natural toxin which exists widely in aquatic environments and has been reported to be hepatotoxic and carcinogenic. At present, the promoting mechanism of MC-LR on hepatocellular carcinoma (HCC) remains largely unexplored. In this study, the hepatocellular promoting effect of MC-LR was described in Kras

Proteomics and phosphoproteomics are robust tools to analyze dynamics of post-transcriptional processes during growth and development. A variety of experimental methods and workflows have been published, but most of them were developed for model plants and have not been adapted to high-throughput platforms. Here, we describe an experimental workflow for proteome and phosphoproteome studies tailored to cereal crop tissues. The workflow consists of two parallel parts that are suitable for analyzing protein/phosphoprotein from total proteins and the microsomal membrane fraction. We present phosphoproteomic data regarding quantification coverage and analytical reproducibility for example preparations from maize root and shoot, wheat leaf, and a microsomal protein preparation from maize leaf. To enable users to adjust for tissue specific requirements, we provide two different methods of protein clean-up: traditional ethanol precipitation (PC) and a recently developed technology termed single-pot, solid-phase-enhanced sample preparation (SP3). Both the PC and SP3 methods are effective in the removal of unwanted substances in total protein crude extracts. In addition, two different methods of phosphopeptide enrichment are presented: a TiO

The aim of the current study is to evaluate the remineralization potential of experimental tricalcium silicate (TCS) paste in comparison with more popular remineralizing agents like silver diamine fluoride potassium iodide (SDF-KI) and casein phosphopeptide amorphous calcium phosphate (CPP-ACP) on early enamel lesions.Forty-five patients in the age range of 15-50 years had early enamel lesions on the buccal surface of molar teeth. The patients were randomly divided into three treatment groups (SDF-KI, CPP-ACP, and TCS group) with 15 patients per group. Lesions were evaluated clinically by DIAGNOdent pen immediately and after 3,6,12, and 24 months of treatment.The study was completed with 45 patients and 92 teeth. Twice-daily application of CPP-ACP and TCS paste showed a significant remineralization effect on early enamel lesions after 24 months (p 0.001). Also, annual application of SDF-KI showed a significant remineralization effect after 24 months (p 0.001). There was a significant difference between (SDF-KI and CPP-ACP) and (SDF-KI and TCS) at the different follow-up periods 3,6,12, and 24 months (p 0.001). Meanwhile, there was no significant difference between CPP-ACP and TCS at the mentioned follow-up periods (p 0.05).TCS showed potential remineralization for early enamel lesions.Experimental TCS is a promising remineralizing agent for management of early enamel lesions.