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Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA-MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction/alkylation, direct trypsinization, and phosphopeptide enrichment by TiO 2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n -dodecyl β-d-maltoside precoated tubes and shortening the digestion time, SOP-Phos is completed within 3-4 h with a 1.4-fold higher identification coverage. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved reproducibility (8%-10% CV). With a sample size-comparable spectral library, SOP-Phos-DIA identified 33,787 ± 670 to 22,070 ± 861 phosphopeptides from 5 to 0.5 μg cell lysate and 30,433 ± 284 to 6,548 ± 21 phosphopeptides from 50,000 to 2,500 cells. Such sensitivity enabled mapping key lung cancer signaling sites, such as EGFR autophosphorylation sites Y1197/Y1172 and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on EGFR-TKI sensitive and resistant cells, revealing the interplay of multipathway Hippo-EGFR-ERBB signaling cascades underlying the mechanistic insight into EGFR-TKI resistance. Overall, SOP-Phos-DIA is an efficient and robust protocol that can be easily adapted in the community for microscale phosphoproteomic analysis.

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to perturbations is crucial for further illuminating the functionality of the phosphoproteome. Here we describe µPhos ('microPhos'), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture and small tissue amounts in <8 h total processing time. By greatly minimizing transfer steps and liquid volumes, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify ~17,000 Class I phosphosites in a human cancer cell line using 20 µg starting material, and confidently localize ~6200 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable. We employ µPhos to study drug- and time-dependent response signatures in a leukemia cell line, and by quantifying 30,000 Class I phosphosites in the mouse brain we reveal distinct spatial kinase activities in subregions of the hippocampal formation., (© 2024. The Author(s).)

Calcium is the most abundant mineral in the human body and is involved in critical physiological and cellular processes. It is essential for the development, maintenance, and integrity of bone tissue throughout life. Identifying new natural food-grade chelating agents to improve calcium uptake is of increasing interest. Casein phosphopeptides (CPPs), highly phosphorylated peptides obtained after enzymatic hydrolysis of caseins, represent promising calcium-chelating candidates. The aim of this study was to investigate, using cell culture models, the ability of a digested milk matrix enriched in CPPs to regulate calcium transport through the intestinal barrier and elucidate the involved mechanisms. To this end, a CPP-preparation underwent in vitro static digestion and was subsequently incubated with an intestinal barrier model to monitor calcium uptake and transport. Our results demonstrated that the digested CPP preparation enhanced the trans -epithelial calcium transport via paracellular pathways and that CPPs, identified by peptidomics, crossed the intestinal barrier in the same time.

Small ORF-encoded peptides (SEPs) are a class of low molecular weight proteins and peptides comprising <100 amino acids with important functions in various life activities. Although the sequence length is short, SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. In this work, we enriched phosphopeptides with IMAC and TiO 2 materials and analyzed the phosphorylated SEPs in Hep3B cells. A total of 24 phosphorylated SEPs were identified, and 11 SEPs were coded by ncRNA. For the sequence analysis, we found that the general characteristics of phosphorylated SEPs are roughly the same as canonical proteins. Besides, two phosphorylation SEPs have the Stathmin family signature 2 motif, which can regulate the microtubule cytoskeleton. Some SEPs have domains or signal peptides, indicating their specific functions and subcellular locations. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of some SEPs. However, only one-fifth of the predicted phosphorylation sites were identified by LC/MS/MS, indicating that many SEP PTMs are hidden in the dark, waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation. SIGNIFICANCE: Small ORF-encoded peptides (SEPs) are important in various life activities. Although the sequence length is short (<100AA), SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. We enriched phosphopeptides and analyzed the phosphorylated SEPs in Hep3B cells. That is the first time to explore the PTM of SPEs systematically. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of SEPs. More SEP PTMs are hidden in the dark and waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phosphorylation is the most studied post-translational modification, and has multiple biological functions. In this study, we have reanalyzed publicly available mass spectrometry proteomics data sets enriched for phosphopeptides from Asian rice ( Oryza sativa ). In total we identified 15,565 phosphosites on serine, threonine, and tyrosine residues on rice proteins. We identified sequence motifs for phosphosites, and link motifs to enrichment of different biological processes, indicating different downstream regulation likely caused by different kinase groups. We cross-referenced phosphosites against the rice 3,000 genomes, to identify single amino acid variations (SAAVs) within or proximal to phosphosites that could cause loss of a site in a given rice variety and clustered the data to identify groups of sites with similar patterns across rice family groups. The data has been loaded into UniProt Knowledge-Base─enabling researchers to visualize sites alongside other data on rice proteins, e.g., structural models from AlphaFold2, PeptideAtlas, and the PRIDE database─enabling visualization of source evidence, including scores and supporting mass spectra.

Summary: Identification and quantification of phosphorylation sites are essential for biological interpretation of a phosphoproteomics experiment. For data independent acquisition mass spectrometry-based (DIA-MS) phosphoproteomics, extracting a site-level report from the output of current processing software is not straightforward as multiple peptides might contribute to a single site, multiple phosphorylation sites can occur on the same peptides, and protein isoforms complicate site specification. Currently only limited support is available from a commercial software package via a platform-specific solution with a rather simple site quantification method. Here, we present sitereport, a software tool implemented in an extendable Python package called msproteomics to report phosphosites and phosphopeptides from a DIA-MS phosphoproteomics experiment with a proven quantification method called MaxLFQ. We demonstrate the use of sitereport for downstream data analysis at site level, allowing benchmarking different DIA-MS processing software tools., Availability and Implementation: sitereport is available as a command line tool in the Python package msproteomics, released under the Apache License 2.0 and available from the Python Package Index (PyPI) at https://pypi.org/project/msproteomics and GitHub at https://github.com/tvpham/msproteomics., (© The Author(s) 2024. Published by Oxford University Press.)

Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 robot that combines sample digestion, cleanup, and loading on Evotips in a fully automated manner, allowing the processing of up to 192 samples in 6 h. Analysis of 192 automated HeLa cell sample preparations consistently identified ∼8000 protein groups and ∼130,000 peptide precursors with an 11.5 min active liquid chromatography gradient with the Evosep One and narrow-window data-independent acquisition (nDIA) with the Orbitrap Astral mass spectrometer providing a throughput of 100 samples per day. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic titanium-immobilized metal ion affinity chromatography beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated digestion and Evotip loading workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients., Competing Interests: Conflict of interest D. B. B.-J., N. B., M. H., and J. M. V.-N. are employees of Evosep Biosystems. The other authors declare no competing interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-bead phosphoproteomics sample preparation with a focus on low-input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores, and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample, and loading buffer volumes allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 μg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA)., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Due to their oftentimes ambiguous nature, phosphopeptide positional isomers can present challenges in bottom-up mass spectrometry-based workflows as search engine scores alone are often not enough to confidently distinguish them. Additional scoring algorithms can remedy this by providing confidence metrics in addition to these search results, reducing ambiguity. Here we describe challenges to interpreting phosphoproteomics data and review several different approaches to determine sites of phosphorylation for both data-dependent and data-independent acquisition-based workflows. Finally, we discuss open questions regarding neutral losses, gas-phase rearrangement, and false localization rate estimation experienced by both types of acquisition workflows and best practices for managing ambiguity in phosphosite determination., (© 2023 The Authors. PROTEOMICS published by Wiley‐VCH GmbH.)

Background: Genetic knockout-based studies conducted in mice provide a powerful means of assessing the significance of a gene for fertility. Forkhead-associated phosphopeptide binding domain 1 (FHAD1) contains a conserved FHA domain, that is present in many proteins with phospho-threonine reader activity. How FHAD1 functions in male fertility, however, remains uncertain., Methods: Fhad1 -/- mice were generated by CRISPR/Cas9-mediated knockout, after which qPCR was used to evaluate changes in gene expression, with subsequent analyses of spermatogenesis and fertility. The testis phenotypes were also examined using immunofluorescence and histological staining, while sperm concentrations and motility were quantified via computer-aided sperm analysis. C ellular apoptosis was assessed using a TUNEL staining assay., Results: The Fhad1 -/- mice did not exhibit any abnormal changes in fertility or testicular morphology compared to wild-type littermates. Histological analyses confirmed that the testicular morphology of both Fhad1 -/- and Fhad1 +/+ mice was normal, with both exhibiting intact seminiferous tubules. Relative to Fhad1 +/+ mice, however, Fhad1 -/- did exhibit reductions in the total and progressive motility of epididymal sperm. Analyses of meiotic division in Fhad1 -/- mice also revealed higher levels of apoptotic death during the first wave of spermatogenesis., Discussion: The findings suggest that FHAD1 is involved in both meiosis and the modulation of sperm motility., Competing Interests: The authors declare that they have no competing interests., (© 2024 Zhang et al.)

The comprehensive investigation of protein phosphorylation and glycosylation aids in the discovery of novel biomarkers as well as the understanding of the pathophysiology of illness. In this work, a nitrogen/titanium-rich porous organic polymer was developed by copolymerizing carbohydrazide (CH) and 2,3-dihydroxyterephthalaldehyde (2,3-Dha) and modifying with Ti 4+ (CH-Dha-Ti 4+ ). The adequate nitrogen contributes to the enrichment of glycopeptides via HILIC, while titanium benefits from capturing phosphopeptides through IMAC. The proposed method exhibits excellent selectivity (1 : 1000, both for glycopeptides and phosphopeptides), LOD (for glycopeptides: 0.05 fmol μL -1 , for phosphopeptides: 0.2 fmol), loading capacity (for glycopeptides: 100 mg g -1 , for phosphopeptides: 125 mg g -1 ) and size-exclusion effect (1 : 10 000, both for glycopeptides and phosphopeptides). Furthermore, CH-Dha-Ti 4+ was applied to capture glycopeptides and phosphopeptides from human serum; 205 glycopeptides and 45 phosphopeptides were detected in the serum of normal controls; and 294 glycopeptides and 63 phosphopeptides were found in the serum of uremia patients after being analyzed by nano LC-MS/MS. The discovered glycopeptides and phosphopeptides were involved in several molecular biological processes and activities, according to a gene ontology study.

Shotgun phosphoproteomics enables high-throughput analysis of phosphopeptides in biological samples. One of the primary challenges associated with this technology is the relatively low rate of phosphopeptide identification during data analysis. This limitation hampers the full realization of the potential offered by shotgun phosphoproteomics. Here we present DeepRescore2, a computational workflow that leverages deep learning-based retention time and fragment ion intensity predictions to improve phosphopeptide identification and phosphosite localization. Using a state-of-the-art computational workflow as a benchmark, DeepRescore2 increases the number of correctly identified peptide-spectrum matches by 17% in a synthetic dataset and identifies 19% to 46% more phosphopeptides in biological datasets. In a liver cancer dataset, 30% of the significantly altered phosphosites between tumor and normal tissues and 60% of the prognosis-associated phosphosites identified from DeepRescore2-processed data could not be identified based on the state-of-the-art workflow. Notably, DeepRescore2-processed data uniquely identifies EGFR hyperactivation as a new target in poor-prognosis liver cancer, which is validated experimentally. Integration of deep learning prediction in DeepRescore2 improves phosphopeptide identification and facilitates biological discoveries., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: B. Z. received consulting fee from AstraZeneca. The remaining author declares no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Calcium is important in the formation of bones and teeth, cell metabolism, and other physiological activities. In this work, casein phosphopeptide-calcium chelate (CPP-Ca) was synthesized and the optimal process parameters for the chelation reaction were obtained. The bioavailability of calcium in CPP-Ca was investigated by in vitro gastrointestinal simulated digestion. The existence of phytic acid and oxalic acid in the digestion system was evaluated to clarify the calcium holding ability of casein phosphopeptide (CPP). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify oligopeptides from CPP-Ca., Results: The optimal process parameters for the chelation reaction were: peptide concentration 7.76 mgmL -1 , pH 8.54, and reaction temperature 43.3 °C. The digestion in vitro results indicated that the calcium release rate of CPP-Ca in the stomach for 2 h reached 85%, and about 50% of the ionized calcium was re-chelated with CPP in the intestine. Phytic acid and oxalic acid could lead to a sharp decrease in soluble calcium but around 50% of the calcium was still retained in the form of chelates in the presence of CPP. The LC-MS/MS identified 19 casein-derived oligopeptides after digestion, and calcium modifications were found on eight peptides derived from β-casein and α s2 -casein., Conclusions: This study clarified the excellent calcium holding capacity of CPP in the presence of phytic acid and oxalic acid. Liquid chromatography-tandem mass spectrometry also revealed peptide changes, and identified peptides that chelate with calcium. These findings provided significant insights that could be relevant to the further utilization and product development of peptide-calcium chelate in the food industry. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Targeted proteomics enables sensitive and specific quantification of proteins and post-translational modifications. By coupling peptide immunoaffinity enrichment with targeted mass spectrometry, we have developed the methodology for multiplexed quantification of proteins and phosphosites involved in the RAS/MAPK signaling network. The method uses anti-peptide antibodies to enrich analytes and heavy stable isotope-labeled internal standards, spiked in at known concentrations. The enriched peptides are directly measured by multiple-reaction monitoring (MRM), a well-characterized quantitative mass spectrometry-based method. The analyte (light) peptide response is measured relative to the heavy standard. The method described provides quantitative measurements of phospho-signaling and is generally applicable to other phosphopeptides and sample types., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein phosphorylation regulates a variety of important cellular and physiological processes in plants. In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphoproteomes. This is largely due to the need to improve protein extraction efficiency from plant cells, which have a dense cell wall, and to minimize sample loss resulting from the stringent sample clean-up steps required for the removal of a large amount of biomolecules interfering with phosphopeptide purification and mass spectrometry analysis. To this end, we developed a method with a streamlined workflow for highly efficient purification of phosphopeptides from tissues of various green organisms including Arabidopsis, rice, tomato, and Chlamydomonas reinhardtii, enabling in-depth identification with high quantitative reproducibility of about 11 000 phosphosites, the greatest depth achieved so far with single liquid chromatography-mass spectrometry (LC-MS) runs operated in a data-dependent acquisition (DDA) mode. The mainstay features of the method are the minimal sample loss achieved through elimination of sample clean-up before protease digestion and of desalting before phosphopeptide enrichment and hence the dramatic increases of time- and cost-effectiveness. The method, named GreenPhos, combined with single-shot LC-MS, enabled in-depth quantitative identification of Arabidopsis phosphoproteins, including differentially phosphorylated spliceosomal proteins, at multiple time points during salt stress and a number of kinase substrate motifs. GreenPhos is expected to serve as a universal method for purification of plant phosphopeptides, which, if samples are further fractionated and analyzed by multiple LC-MS runs, could enable measurement of plant phosphoproteomes with an unprecedented depth using a given mass spectrometry technology., (Copyright © 2023 The Author. Published by Elsevier Inc. All rights reserved.)

Calcium is one of the important elements for human health. Calcium deficiencies can lead to numerous diseases. Calcium chelating peptides have shown potential application in the management of calcium deficiencies. Casein phosphopeptides (CPP) are phosphoseryl-containing fragments of casein by enzymatic hydrolysis or fermentation during manufacture of milk products as well as during intestinal digestion. An increasing number of CPP with the ability to facilitate and enhance the bioavailability of calcium are being discovered and identified. In this review, 249 reported CPP derived from four types of bovine casein (αs1, αs2, β and κ) were collected, and the amino acid sequence and phosphoserine group information were sorted out. This review outlines the current enzyme hydrolysis, detection methods, purification, structure-activity relationship and mechanism of intestinal calcium absorption in vitro and in vivo as well as application of CPP.

Our previous studies have shown that highly phosphorylated casein phosphopeptides (residues 1-25) P5 could efficiently bind calcium and promote intestinal calcium absorption, and enhanced bone development in rats. The purpose of this study was to investigate the effect of the phosphorylation structure in P5 on the proliferation, differentiation, and mineralization of osteoblasts (MC3T3-E1) and its mechanism. P5 was obtained by high-performance liquid chromatography (HPLC) and non-phosphorylated peptide P5-0 was obtained by chemical synthesis. Compared with the control group, the proliferation rate of MC3T3-E1 cells treated by P5 was 1.10 times that of P5-0 at 200 μg mL -1 . P5 caused the cell cycle retention of MC3T3-E1 cells in the G2/M phase, while P5-0 had no significant difference in the G2/M phase. MC3T3-E1 cells incubated with P5 showed stronger alkaline phosphatase (ALP) activity than with P5-0, suggesting a tendency to promote cellular differentiation. Compared to the P5-0 treatment group, the P5 treatment group at concentrations of 10 μg mL -1 showed significant differences in the mineralization rates ( p < 0.05). P5 significantly upregulated the expressions of Runx2, ALP, ColIα1, and OCN compared with the control group ( p < 0.05). In addition, in silico molecular docking showed that the binding force of the P5-EGFR complex was stronger than that of the P5-0-EGFR complex, which was significantly related to the phosphorylation structure in P5 and might be an important reason for osteoblast proliferation. In conclusion, the phosphorylation structure and amino acid composition in P5 stimulated the osteogenic activity of MC3T3-E1 cells, and could be expected to be a functional food for the prevention of osteoporosis.

Sodium bicarbonate stress caused by NaHCO 3 is one of the most severe abiotic stresses affecting agricultural production worldwide. However, little attention has been given to the molecular mechanisms underlying plant responses to sodium bicarbonate stress. To understand phosphorylation events in signaling pathways triggered by sodium bicarbonate stress, TMT-labeling-based quantitative phosphoproteomic analyses were performed on soybean leaf and root tissues under 50 mM NaHCO 3 treatment. In the present study, a total of 7856 phosphopeptides were identified from cultivated soybeans ( Glycine max L. Merr.), representing 3468 phosphoprotein groups, in which 2427 phosphoprotein groups were newly identified. These phosphoprotein groups contained 6326 unique high-probability phosphosites (UHPs), of which 77.2% were newly identified, increasing the current soybean phosphosite database size by 43.4%. Among the phosphopeptides found in this study, we determined 67 phosphopeptides (representing 63 phosphoprotein groups) from leaf tissue and 554 phosphopeptides (representing 487 phosphoprotein groups) from root tissue that showed significant changes in phosphorylation levels under sodium bicarbonate stress (fold change >1.2 or <0.83, respectively; p < 0.05). Localization prediction showed that most phosphoproteins localized in the nucleus for both leaf and root tissues. GO and KEGG enrichment analyses showed quite different enriched functional terms between leaf and root tissues, and more pathways were enriched in the root tissue than in the leaf tissue. Moreover, a total of 53 different protein kinases and 7 protein phosphatases were identified from the differentially expressed phosphoproteins (DEPs). A protein kinase/phosphatase interactor analysis showed that the interacting proteins were mainly involved in/with transporters/membrane trafficking, transcriptional level regulation, protein level regulation, signaling/stress response, and miscellaneous functions. The results presented in this study reveal insights into the function of post-translational modification in plant responses to sodium bicarbonate stress.

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

There is growing interest in the development of materials for enriching proteins and phosphoproteins from complex sample matrices for mass spectrometric analysis. Herein, we designed and synthesized two types of magnetic resin composites, i.e. , MTS9200@Fe 3 O 4 and FPA90CL@Fe 3 O 4 , and assessed their applications as adsorbents for enriching proteins, peptides and phosphopeptides. With the combination of Fe 3+ -IMAC interaction (MTS9200) or electrostatic attraction (FPA90CL) of resins and the adsorption of Fe 3 O 4 , the prepared composites exhibited higher capacities for adsorbing a protein (bovine serum albumin, at 195.71 and 135.03 mg g -1 for MTS9200@Fe 3 O 4 and FPA90CL@Fe 3 O 4 , respectively) than MTS9200, FPA90CL and Fe 3 O 4 . In addition, due to the contributions of the hydrophobic skeleton of resins and Fe 3 O 4 , the magnetic resin composites allowed for efficient enrichment of peptides. Moreover, through Fe 3+ -IMAC interaction or electrostatic attraction of resins and Fe-O MOAC interaction of Fe 3 O 4 with phosphate groups, phosphopeptides could also be captured. Furthermore, we employed the prepared composites for enriching proteins and phosphopeptides from human serum, where 466 and 506 proteins, and 434 and 356 phosphorylation sites, were detected from human serum after being processed with FPA90CL@Fe 3 O 4 and MTS9200@Fe 3 O 4 , respectively. Together, our work revealed the great potential of magnetic resin composites as enrichment materials for proteomics and phosphoproteomics analysis.

Human papillomaviruses (HPVs) can increase the proliferation of infected cells during HPV-driven abnormalities, such as cervical cancer or benign warts. To date, more than 200 HPV genotypes have been identified, most of which are classified into three major genera: Alphapapillomavirus, Betapapillomavirus, and Gammapapillomavirus. HPV genomes commonly encode two structural (L1 and L2) and seven functional (E1, E2, E4-E7, and E8) proteins. L2, the minor structural protein of HPVs, not only serves as a viral capsid component but also interacts with various human proteins during viral infection. A recent report revealed that L2 of HPV16 recruits polo-like kinase 1 (Plk1), a master regulator of eukaryotic mitosis and cell cycle progression, for the delivery of viral DNA to mitotic chromatin during HPV16 infection. In this study, we verified the direct and potent interactions between the polo-box domain (PBD) of Plk1 and PBD-binding motif (S-S-pT-P)-containing phosphopeptides derived from L2 of HPV16/HPV18 (high-risk alphapapillomaviruses), HPV5b (low-risk betapapillomavirus), and HPV4 (low-risk gammapapillomavirus). Subsequent structural determination of the Plk1 PBD bound to the HPV18 or HPV4 L2-derived phosphopeptide demonstrated that they interact with each other in a canonical manner, in which electrostatic interactions and hydrogen bonds play key roles in sustaining the complex. Therefore, our structural and biochemical data imply that Plk1 is a broad binding target of L2 of various HPV genotypes belonging to the Alpha-, Beta-, and Gammapapillomavirus genera., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Many biological processes are regulated through dynamic protein phosphorylation. Monitoring disease-relevant phosphorylation events in circulating biofluids is highly appealing but also technically challenging. We introduce here a functionally tunable material and a strategy, extracellular vesicles to phosphoproteins (EVTOP), which achieves one-pot extracellular vesicles (EVs) isolation, extraction, and digestion of EV proteins, and enrichment of phosphopeptides, with only a trace amount of starting biofluids. EVs are efficiently isolated by magnetic beads functionalized with Ti IV ions and a membrane-penetrating peptide, octa-arginine R 8 + , which also provides the hydrophilic surface to retain EV proteins during lysis. Subsequent on-bead digestion concurrently converts EVTOP to Ti IV ion-only surface for efficient enrichment of phosphopeptides for phosphoproteomic analyses. The streamlined, ultra-sensitive platform enabled us to quantify 500 unique EV phosphopeptides with only a few μL of plasma and over 1200 phosphopeptides with 100 μL of cerebrospinal fluid (CSF). We explored its clinical application of monitoring the outcome of chemotherapy of primary central nervous system lymphoma (PCNSL) patients with a small volume of CSF, presenting a powerful tool for broad clinical applications., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Introduction: Protein phosphorylation is a critical post-translational modification involved in the regulation of numerous cellular processes from signal transduction to modulation of enzyme activities. Knowledge of dynamic changes of phosphorylation levels during biological processes, under various treatments or between healthy and disease models is fundamental for understanding the role of each phosphorylation event. Thereby, LC-MS/MS based technologies in combination with quantitative proteomics strategies evolved as a powerful strategy to investigate the function of individual protein phosphorylation events., Areas Covered: State-of-the-art labeling techniques including stable isotope and isobaric labeling provide precise and accurate quantification of phosphorylation events. Here, we review the strengths and limitations of recent quantification methods and provide examples based on current studies, how quantitative phosphoproteomics can be further optimized for enhanced analytic depth, dynamic range, site localization, and data integrity. Specifically, reducing the input material demands is key to a broader implementation of quantitative phosphoproteomics, not least for clinical samples., Expert Opinion: Despite quantitative phosphoproteomics is one of the most thriving fields in the proteomics world, many challenges still have to be overcome to facilitate even deeper and more comprehensive analyses as required in the current research, especially at single cell levels and in clinical diagnostics.

Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains unclear. Here we describe a mechanism by which an HLA-B*0702-specific acute myeloid leukemia phosphoneoantigen, pMLL 747-755 (EPR(pS)PSHSM), is recognized by a cognate T cell receptor named TCR27, a candidate for cancer immunotherapy. We show that the replacement of phosphoserine P 4 with serine or phosphomimetics does not affect pMHC conformation or peptide-MHC affinity but abrogates TCR27-dependent T cell activation and weakens binding between TCR27 and pMHC. Here we describe the crystal structures for TCR27 and cognate pMHC, map of the interface produced by nuclear magnetic resonance, and a ternary complex generated using information-driven protein docking. Our data show that non-covalent interactions between the epitope phosphate group and TCR27 are crucial for TCR specificity. This study supports development of new treatment options for cancer patients through target expansion and TCR optimization., (© 2023. The Author(s).)

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.e., SureQuant) using EGF-stimulated HeLa cells and find the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiles the global phosphoproteome. To demonstrate the robustness, sensitivity, and biomedical potential of hybrid-DIA, we profile chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluate the phospho-signaling difference of cancer cells in 2D vs 3D culture., (© 2023. The Author(s).)

The two non-visual arrestins, arrestin2 and arrestin3, bind hundreds of GPCRs with different phosphorylation patterns, leading to distinct functional outcomes. Structural information on these interactions is available only for very few GPCRs. Here, we have characterized the interactions between the phosphorylated human CC chemokine receptor 5 (CCR5) and arrestin2. We identified several new CCR5 phosphorylation sites necessary for stable arrestin2 complex formation. Structures of arrestin2 in the apo form and complexes with CCR5 C-terminal phosphopeptides, together with NMR, biochemical, and functional assays, revealed three phosphoresidues in a pXpp motif that are essential for arrestin2 binding and activation. The identified motif appears responsible for robust arrestin2 recruitment in many other GPCRs. An analysis of receptor sequences and available structural and functional information provides hints on the molecular basis of arrestin2/arrestin3 isoform specificity. Our findings demonstrate how multi-site phosphorylation controls GPCR⋅arrestin interactions and provide a framework to probe the intricate details of arrestin signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Posttranslational modifications of proteins play essential roles in defining and regulating the functions of the proteins they decorate, making identification of these modifications critical to understanding biology and disease. Methods for enriching and analyzing a wide variety of biological and chemical modifications of proteins have been developed using mass spectrometry-based proteomics, largely relying on traditional database search methods to identify the resulting mass spectra of modified peptides. These database search methods treat modifications as static attachments of a mass to particular position in the peptide sequence, but many modifications undergo fragmentation in tandem mass spectrometry experiments alongside, or instead of, the peptide backbone. While this fragmentation can confound traditional search methods, it also offers unique opportunities for improved searches that incorporate modification-specific fragment ions. Here, we present a new labile mode in the MSFragger search engine that provides the flexibility to tailor modification-centric searches to the fragmentation observed. We show that labile mode can dramatically improve spectrum identification rates of phosphopeptides, RNA-crosslinked peptides, and ADP-ribosylated peptides. Each of these modifications presents distinct fragmentation characteristics, showcasing the flexibility of MSFragger labile mode to improve search for a wide variety of biological and chemical modifications., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein conformational changes with fluctuations are fundamental aspects of protein-protein interactions (PPIs); understanding these motions is required for the rational design of PPI-regulating compounds. Src homology 2 (SH2) domains are commonly found in adapter proteins involved in signal transduction and specifically bind to consensus motifs of proteins containing phosphorylated tyrosine (pY). Here, we analysed the interaction between the N-terminal SH2 domain (nSH2) of the regulatory subunit in phosphoinositide 3-kinase (PI3K) and the cytoplasmic region of the T-cell co-receptor, CD28, using NMR and molecular dynamics (MD) simulations. First, we assigned the backbone signals of nSH2 on 1 H- 15 N heteronuclear single quantum coherence spectra in the absence or presence of the CD28 phosphopeptide, SDpYMNMTPRRPG. Chemical shift perturbation experiments revealed allosteric changes at the BC loop and the C-terminal region of nSH2 upon CD28 binding. NMR relaxation experiments showed a conformational exchange associated with CD28 binding in these regions. The conformational stabilisation of the C-terminal region correlated with the regulation of PI3K catalytic function. Further, using 19 F- and 31 P-labelled CD28 phosphopeptide, we analysed the structural dynamics of CD28 and demonstrated that the aromatic ring of the pY residue fluctuated between multiple conformations upon nSH2 binding. Our MD simulations largely explained the NMR results and the structural dynamics of nSH2 and CD28 in both bound and unbound states. Notably, in addition to its major conformation, we detected a minor conformation of nSH2 in the CD28 bound state that may explain the allosteric conformational change in the BC loop., (© 2022 Federation of European Biochemical Societies.)

Protein phosphorylation is a post-translational modification crucial for many cellular processes and protein functions. Accurate identification and quantification of protein phosphosites at the proteome-wide level are challenging, not least because efficient tools for protein phosphosite false localization rate (FLR) control are lacking. Here, we propose DeepFLR, a deep learning-based framework for controlling the FLR in phosphoproteomics. DeepFLR includes a phosphopeptide tandem mass spectrum (MS/MS) prediction module based on deep learning and an FLR assessment module based on a target-decoy approach. DeepFLR improves the accuracy of phosphopeptide MS/MS prediction compared to existing tools. Furthermore, DeepFLR estimates FLR accurately for both synthetic and biological datasets, and localizes more phosphosites than probability-based methods. DeepFLR is compatible with data from different organisms, instruments types, and both data-dependent and data-independent acquisition approaches, thus enabling FLR estimation for a broad range of phosphoproteomics experiments., (© 2023. The Author(s).)

Innovative methods for engineering cancer cell membranes promise to manipulate cell-cell interactions and boost cell-based cancer therapeutics. Here, we illustrate an in situ approach to selectively modify cancer cell membranes by employing an enzyme-instructed peptide self-assembly (EISA) strategy. Using three phosphopeptides ( pY1 , pY2 , and pY3 ) targeting the membrane-bound epidermal growth factor receptor (EGFR) and differing in just one phosphorylated tyrosine, we reveal that site-specific phosphorylation patterns in pY1 , pY2 , and pY3 can distinctly command their preorganization levels, self-assembling kinetics, and spatial distributions of the resultant peptide assemblies in cellulo . Overall, pY1 is the most capable of producing preorganized assemblies and shows the fastest dephosphorylation reaction in the presence of alkaline phosphatase (ALP), as well as the highest binding affinity for EGFR after dephosphorylation. Consequently, pY1 exhibits the greatest capacity to construct stable peptide assemblies on cancer cell membranes with the assistance of both ALP and EGFR. We further use peptide-protein and peptide-peptide co-assembly strategies to apply two types of antigens, namely ovalbumin (OVA) protein and dinitrophenyl (DNP) hapten respectively, on cancer cell membranes. This study demonstrates a very useful technique for the in situ construction of membrane-bound peptide assemblies around cancer cells and implies a versatile strategy to artificially enrich cancer cell membrane components for potential cancer immunotherapy.

Protein phosphorylation is one of the most ubiquitous post-translational modifications, regulating numerous essential processes in cells. Accordingly, the large-scale annotation of phosphorylation sites continues to provide central insight into the regulation of signaling networks. The global analysis of the phosphoproteome typically relies on mass spectrometry analysis of phosphopeptides, with an enrichment step necessary due to the sub-stoichiometric nature of phosphorylation. Several affinity-based methods and chemical modification strategies have been developed to date, but the choice of enrichment method can have a considerable impact on the results. Here, we show that a biotinylated, photo-cleavable phosphorimidazolide reagent permits the immobilization and subsequent cleavage of phosphopeptides. The method is capable of the capture and release of phosphopeptides of varying characteristics, and this mild and selective strategy expands the current repertoire for phosphopeptide chemical modification with the potential to enrich and identify new phosphorylation sites in the future., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Background: Extensive evidence from single-center studies indicates that a subset of patients with chronic advanced heart failure (HF) undergoing left ventricular assist device (LVAD) support show significantly improved heart function and reverse structural remodeling (ie, termed "responders"). Furthermore, we recently published a multicenter prospective study, RESTAGE-HF (Remission from Stage D Heart Failure), demonstrating that LVAD support combined with standard HF medications induced remarkable cardiac structural and functional improvement, leading to high rates of LVAD weaning and excellent long-term outcomes. This intriguing phenomenon provides great translational and clinical promise, although the underlying molecular mechanisms driving this recovery are largely unknown., Methods: To identify changes in signaling pathways operative in the normal and failing human heart and to molecularly characterize patients who respond favorably to LVAD unloading, we performed global RNA sequencing and phosphopeptide profiling of left ventricular tissue from 93 patients with HF undergoing LVAD implantation (25 responders and 68 nonresponders) and 12 nonfailing donor hearts. Patients were prospectively monitored through echocardiography to characterize their myocardial structure and function and identify responders and nonresponders., Results: These analyses identified 1341 transcripts and 288 phosphopeptides that are differentially regulated in cardiac tissue from nonfailing control samples and patients with HF. In addition, these unbiased molecular profiles identified a unique signature of 29 transcripts and 93 phosphopeptides in patients with HF that distinguished responders after LVAD unloading. Further analyses of these macromolecules highlighted differential regulation in 2 key pathways: cell cycle regulation and extracellular matrix/focal adhesions., Conclusions: This is the first study to characterize changes in the nonfailing and failing human heart by integrating multiple -omics platforms to identify molecular indices defining patients capable of myocardial recovery. These findings may guide patient selection for advanced HF therapies and identify new HF therapeutic targets.

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 TiO 2 in a batch that enables measurement of protein posttranslational modifications is a powerful application to analyze the global phosphoproteome for studies in signaling pathways., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Inflammation in early life is a risk factor for the development of neuropsychiatric diseases later in adolescence and adulthood, yet the underlying mechanism remains elusive. In the present study, we performed an integrated proteomic and phosphoproteomic analysis of the hippocampus to identify potential molecular mechanisms of early life inflammation-induced cognitive impairment., Methods: Both female and male mice received a single intraperitoneal injection of 100 μg/kg lipopolysaccharide (LPS) on postnatal day 10 (P10). Behavioral tests, including open field, elevated plus-maze, and Y-maze tests, were performed on P39, P40, and P41, respectively. After behavioral tests, male mice were sacrificed. The whole brain tissues and the hippocampi were harvested on P42 for proteomic, phosphoproteomic, Western blot, and Golgi staining., Results: Early life LPS exposure induced cognitive impairment in male mice but not in female mice, as assessed by the Y-maze test. Therefore, following biochemical tests were conducted on male mice. By proteomic analysis, 13 proteins in LPS group exhibited differential expression. Among these, 9 proteins were upregulated and 4 proteins were downregulated. For phosphoproteomic analysis, a total of 518 phosphopeptides were identified, of which 316 phosphopeptides were upregulated and 202 phosphopeptides were downregulated in the LPS group compared with the control group. Furthermore, KEGG analysis indicated that early life LPS exposure affected the glutamatergic synapse and neuroactive ligand-receptor interaction, which were associated with synaptic function and energy metabolism. Increased level of brain protein i3 (Bri3), decreased levels of PSD-95 and mGLUR5, and dendritic spine loss after early life LPS exposure further confirmed the findings of proteomic and phosphoproteomic analysis., Conclusions: Our findings demonstrated that neuroinflammation and impaired synapse may be involved in early life inflammation-induced cognitive impairment. Future studies are required to confirm our preliminary results., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Abnormally hyperphosphorylated tau can be recognized by a variety of phosphoprotein-binding domains (PBDs) to elicit downstream tau signaling in neuropathology, which has been found to have a potential association with subarachnoid hemorrhage. In this study, the genome-wide binding behavior of tau phosphorylation sites (p-sites) to PBDs involved in subarachnoid hyperphosphorylation events was systematically profiled at molecular level by integrating peptide docking, structural minimization, affinity scoring, and binding assay, from which a number of potent PBD-p-site interaction pairs were identified. It was revealed that the PBD domains exhibit distinct binding preferences for phosphotyrosine, phosphoserine, and phosphothreonine p-sites; the PBD-recognition specificity of different tau p-sites is not overlapped with each other, and their phosphorylations would therefore regulate varying biological functions in tau signaling. A number of PBD-p-site pairs were identified to have potent binding potency as compared to others. The KCIP-pS[393-399] pair was found as a strong binder, which was further optimized with a rational peptide design protocol to derive a number of affinity-improved phosphopeptides. Structural analysis revealed diverse noncovalent chemical forces across the complex interface of KCIP domain with a designed high-affinity pS[393-399]-d4, which confers both stability and specificity to the domain-peptide complex system, with affinity improved by 10.9-fold relative to the native pS[393-399]., (© 2021 International Union of Biochemistry and Molecular Biology, Inc.)

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., (© 2022 Wiley-VCH GmbH.)

The small intestine is the primary site of nutrient digestion and absorption, which plays a key role in the survival of neonatal calves. A comprehensive assessment of the phosphoproteomic changes in the small intestine of neonatal calves is unavailable; therefore, we used phosphopeptide enrichment coupled with liquid chromatography-tandem mass spectrometry to investigate the changes in the phosphoproteome profile in the bovine small intestine during the first 36 h of life. Twelve neonatal male calves were assigned to one of the following groups: (1) calves not fed colostrum and slaughtered approximately 2 h postpartum (n = 3), (2) calves fed colostrum at 1 to 2 h and slaughtered 8 h postpartum (n = 3), (3) calves fed 2 colostrum meals (at 1-2 and 10-12 h) and slaughtered 24 h postpartum (n = 3), (4) calves fed 3 colostrum meals (at 1-2, 10-12, and 22-24 h) and slaughtered 36 h postpartum (n = 3). Mid-duodenal, jejunal, and ileal samples of the calves were collected after slaughter. We identified 1,678 phosphoproteins with approximately 3,080 phosphosites, which were mainly Ser (89.9%), Thr (9.8%), and Tyr (0.3%) residues; they belonged to the prodirected (52.9%), basic (20.4%), acidic (16.6%), and Tyr-directed (1.7%) motif categories. The regional differentially expressed phosphoproteins included zonula occludens 2, sorting nexin 12, and protein kinase C, which are mainly associated with developmental processes, intracellular transport, vesicle-mediated transport, and immune system process. They are enriched in the endocytosis, tight junction, insulin signaling, and focal adhesion pathways. The temporal differentially expressed phosphoproteins included occludin, epsin 1, and bridging integrator 1, which were mainly associated with macromolecule metabolic process, cell adhesion, and growth. They were enriched in the spliceosomes, adherens junctions, and tight junctions. The observed changes in the phosphoproteins in the tissues of small intestine suggest the protein phosphorylation plays an important role in nutrient transport and immune response of calves during early life, which needs to be confirmed in a larger study., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Objective: Osteoarthritis (OA) causes gradual cellular alterations, structural anomalies and joint dysfunction. Progressive decline of chondrocyte function plays a vital role on OA pathogenesis. Although protein phosphorylation controls cartilage metabolism, its regulation mechanism in OA remains unclear. Thus, proteomic methods were used to investigate phosphorylation changes in preserved and OA articular cartilage samples, and to explore the intervention targets of phosphorylated kinase., Methods: Preserved (control) and lesioned (OA) cartilage samples from OA cases were assessed by phosphoproteomics. Immobilized metal affinity chromatography was performed for phosphopeptide enrichment. Quantitated phosphosites were comparatively assessed in the cartilage sample pair. Kinase-substrate enrichment analyses were carried out for identifying OA-related kinases. BRAF expression in cartilage tissues was assessed by immunohistochemical staining. The effects of BRAF inhibitor on cartilage degeneration were examined in mouse chondrocytes and OA mouse model., Results: High-sensitivity mass spectrometry-based proteomics revealed 7,471 peptides and 4,375 phosphorylated peptides differing between preserved and lesioned cartilage samples, which represented the significant alteration of kinase hubs and transduction pathways. Phosphoproteomics identified BRAF may be involved in developing OA. BRAF regulated the downstream ERK signaling pathway. In addition, BRAF was upregulated in human OA cartilage as shown by immunohistochemistry. Remarkably, BRAF inhibition alleviated cartilage degradation in a mouse model of OA through its downstream of ERK pathway activation., Conclusions: Jointly, these findings provide an overview of phosphoproteomic alterations occurring during cartilage degeneration, identifying the BRAF-ERK1/2 Axis signaling as a potential signaling pathway involved in OA., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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 V12 transgenic zebrafish, a doxycycline (DOX) inducible HCC model. Our results showed that MC-LR could aggravate the progression of HCC at an environmentally relevant concentration (3 μg/L), which was accompanied by the decreased activity and down-regulated transcription level of serine/threonine phosphatase 2A (PP2A). Using TMT labeling quantitative phosphoproteomics, we found that the 1049 phosphopeptides were significantly changed (508 up-regulated and 541 down-regulated) in liver from combined exposure to DOX and 3 μg/L MC-LR group compared to the DOX group. Enriched pathways by KEGG analysis suggested that differentially phosphorylated proteins were mainly related to Wnt signaling pathway. Furthermore, the mRNA expression and protein abundance of β-Catenin in Wnt signaling pathway were significantly up-regulated following exposure to MC-LR. In short, our results suggested that MC-LR significantly inhibited the activity of PP2A, which in turn activated Wnt signaling, eventually resulting in progression of liver tumor in transgenic zebrafish., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Protein 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. C57BL/6J female mice (6 mo) were 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, noncontracting condition. After 16 wk of estrogen deficiency, the tibialis anterior muscle was dissected and prepped for label-free nano-liquid chromatography-tandem mass spectrometry phosphoproteomic analysis. 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 < 0.05 and 1.5-fold change ratio) that corresponded to 130 proteins with 22 proteins differentially phosphorylated (3 unique to Ovx, 2 unique to Sham, 6 upregulated, and 11 downregulated). Differentially phosphorylated proteins associated with the sarcomere, cytoplasm, and metabolic and calcium signaling pathways were identified. Our work provides the first global phosphoproteomic analysis in females and how estrogen deficiency impacts the skeletal muscle phosphoproteome.

The diterpene glucoside fusicoccin-A (FC-A) is a fungal phytotoxin that stabilizes the interaction of plant 14-3-3 protein and plasma membrane H + -ATPase by forming a stable ternary complex. Previous studies demonstrated that structurally modified FC-A derivatives exhibit significant antitumor activities but their synthesis involves an explosive reagent, limiting their utility and opportunities for further structure-activity-relationship studies. In this study, we synthesized a series of FC derivatives by introducing various substituents on the fusicoccan scaffold and on the glucoside moiety, and evaluated their stabilization effects on the binding of 14-3-3 to fluorescently labeled mode-1 and mode-3 phosphopeptides. The results showed that introducing an amino group at the 6'-position of the glucoside moiety improves stabilization. Furthermore, cell-based evaluation demonstrated that 6'-amino benzyl 21b exhibits higher antiproliferative activity than previously developed FC agents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mass-spectrometry-based phosphoproteomics has become indispensable for understanding cellular signaling in complex biological systems. Despite the central role of protein phosphorylation, the field still lacks inexpensive, regenerable, and diverse phosphopeptides with ground-truth phosphorylation positions. Here, we present Iterative Synthetically Phosphorylated Isomers (iSPI), a proteome-scale library of human-derived phosphoserine-containing phosphopeptides that is inexpensive, regenerable, and diverse, with precisely known positions of phosphorylation. We demonstrate possible uses of iSPI, including use as a phosphopeptide standard, a tool to evaluate and optimize phosphorylation-site localization algorithms, and a benchmark to compare performance across data analysis pipelines. We also present AScorePro, an updated version of the AScore algorithm specifically optimized for phosphorylation-site localization in higher energy fragmentation spectra, and the FLR viewer, a web tool for phosphorylation-site localization, to enable community use of the iSPI resource. iSPI and its associated data constitute a useful, multi-purpose resource for the phosphoproteomics community., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Clear cell Renal Cell Carcinoma (ccRCC) is among the 10 most common cancers in both men and women and causes more than 140,000 deaths worldwide every year. In order to elucidate the underlying molecular mechanisms orchestrated by phosphorylation modifications, we performed a comprehensive quantitative phosphoproteomics characterization of ccRCC tumor and normal adjacent tissues. Here, we identified 16,253 phosphopeptides, of which more than 9000 were singly quantified. Our in-depth analysis revealed 600 phosphopeptides to be significantly differentially regulated between tumor and normal tissues. Moreover, our data revealed that significantly up-regulated phosphoproteins are associated with protein synthesis and cytoskeletal re-organization which suggests proliferative and migratory behavior of renal tumors. This is supported by a mesenchymal profile of ccRCC phosphorylation events. Our rigorous characterization of the renal phosphoproteome also suggests that both epidermal growth factor receptor and vascular endothelial growth factor receptor are important mediators of phospho signaling in RCC pathogenesis. Furthermore, we determined the kinases p21-activated kinase 2, cyclin-dependent kinase 1 and c-Jun N-terminal kinase 1 to be master kinases that are responsible for phosphorylation of many substrates associated with cell proliferation, inflammation and migration. Moreover, high expression of p21-activated kinase 2 is associated with worse survival outcome of ccRCC patients. These master kinases are targetable by inhibitory drugs such as fostamatinib, minocycline, tamoxifen and bosutinib which can serve as novel therapeutic agents for ccRCC treatment., Competing Interests: Conflict of Interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Tuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis 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 posttranslational arginine phosphorylation. While several lines of evidence suggest that ClpC1P1P2 is similarly capable of recognizing phosphoarginine-bearing proteins, the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of posttranslational phosphoarginine modifications in Mycolicibacterium smegmatis, a nonpathogenic surrogate of M. tuberculosis. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identified arginine phosphosites on several functionally diverse targets within the M. smegmatis proteome. Interestingly, phosphoarginine modifications are not upregulated by heat stress, suggesting divergent roles in mycobacteria and Bacillus . Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species. IMPORTANCE Mycobacteria that cause tuberculosis infections employ proteolytic pathways that modulate cellular behavior by destroying specific proteins in a highly regulated manner. Some proteolytic enzymes have emerged as novel antibacterial targets against drug-resistant tuberculosis infections. However, we have only a limited understanding of how these enzymes function in the cell and how they select proteins for destruction. Some proteolytic enzymes are capable of recognizing proteins that carry an unusual chemical modification, arginine phosphorylation. Here, we confirm the existence of arginine phosphorylation in mycobacterial proteins. Our work expands our understanding of a promising drug target in an important global pathogen.

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.

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 in vivo potency is urgently needed. Here, we report the feasibility of targeting the STAT3-SH2 domain with a boronic acid group and the identification of a highly potent inhibitor compound 7 by replacing the carboxylic acid of compound 4 with a boronic acid. Compound 7 shows higher binding affinity, better cellular potency, more favorable PK profiles, and higher in vivo antitumor activity than 4 . The stronger anticancer effect of 7 partially stems from its covalent binding mode with the SH2 domain, verified by the washout experiments. The relatively high level of sequence conservation among SH2 domains makes the results presented here of general significance.

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., (© 2022. The Author(s).)

Keratoconus (KC) is non-inflammatory, bilateral progressive corneal ectasia, and a disease of established biomechanical instability. The etiology of KC is believed to be multifactorial. Although previous studies gained insight into the understanding of the disease, little is known thus far on global protein phosphorylation changes in keratoconus. We performed phosphoproteome analysis of corneal epithelium from control (N = 5) and KC patients. Tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography (IMAC) were used for the phosphopeptides enrichment and quantitation. Enriched peptides were analyzed on Orbitrap Fusion Tribrid mass spectrometer. This leads to the identification of 2939 unique phosphopeptides derived from 1270 proteins. We observed significant differential phosphorylation of 591 phosphopeptides corresponding to 375 proteins. Our results provide first phosphoproteomic signature of the keratoconus disease and identified dysregulated signaling pathways that can be targeted for therapy in future studies., (© 2022 Wiley-VCH GmbH.)

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is a human granulocyte receptor mediating the efficient phagocytosis of a subset of human-restricted bacterial pathogens. Its function depends on phosphorylation of a tyrosine-based sequence motif, but the enzyme(s) responsible for reversing this modification are unclear. Here, we identify the receptor-type protein tyrosine phosphatase PTPRJ as a negative regulator of CEACAM3-mediated phagocytosis. We show depletion of PTPRJ results in a gain-of-function phenotype, while overexpression of a constitutively active PTPRJ phosphatase strongly reduces bacterial uptake via CEACAM3. We also determined that recombinant PTPRJ directly dephosphorylates the cytoplasmic tyrosine residues of purified full-length CEACAM3 and recognizes synthetic CEACAM3-derived phosphopeptides as substrates. Dephosphorylation of CEACAM3 by PTPRJ is also observed in intact cells, thereby limiting receptor-initiated cytoskeletal re-arrangements, lamellipodia formation, and bacterial uptake. Finally, we show that human phagocytes deficient for PTPRJ exhibit exaggerated lamellipodia formation and enhanced opsonin-independent phagocytosis of CEACAM3-binding bacteria. Taken together, our results highlight PTPRJ as a bona fide negative regulator of CEACAM3-initiated phagocyte functions, revealing a potential molecular target to limit CEACAM3-driven inflammatory responses., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)