Quantitative Phosphoproteomic Analysis Research Articles

Phosphopeptide Enrichment and LC-MS/MS Analysis to Study the Phosphoproteome of Recombinant Chinese Hamster Ovary Cells.

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is one of the most important post-translational modifications that regulates many biological processes. There have been relatively few studies on the phosphoproteome of recombinant Chinese hamster ovary (CHO) cells to date despite phosphorylation playing a crucial role in regulating many molecular and cellular processes relevant to bioprocess phenotypes including, for example, transcription, translation, growth, apoptosis, and signal transduction. In this chapter, we provide a protocol for phosphoproteomic analysis of CHO cells using phosphopeptide enrichment with metal oxide affinity chromatography (MOAC) and immobilized metal affinity chromatography (IMAC) techniques, followed by site-specific identification of phosphorylated residues using liquid chromatography mass spectrometry (LC-MS), multistage activation (MSA), and MS3 strategies. This protocol can also be used for quantitative phosphoproteomic analysis using both labeled and label-free approaches.

Characterization of Ksg1 protein kinase-dependent phosphoproteome in the fission yeast S. pombe

Ksg1 is an essential protein kinase of the fission yeast S. pombe that belongs to the AGC kinase family and is homologous to the mammalian PDPK1 kinase. Previous studies have shown that Ksg1 functions in the nutrient-sensing TOR signaling pathway and is involved in the phosphorylation and activation of other AGC kinases, thereby affecting various downstream targets related to metabolism, cell division, stress response, and gene expression. To date, the molecular function of Ksg1 has been analyzed using its temperature sensitive mutants or mutants expressing its truncated isoforms, which are not always suitable for functional studies of Ksg1 and the identification of its targets. To overcome these limitations, we employed a chemical genetic strategy and used a conditional ksg1as mutant sensitive to an ATP analog. Combining this mutant with quantitative phosphoproteomics analysis, we identified 1986 phosphosites that were differentially phosphorylated when Ksg1as kinase was inhibited by an ATP analog. We found that proteins whose phosphorylation was dysregulated after inhibition of Ksg1as kinase were mainly represented by those involved in the regulation of cytokinesis, contractile ring contraction, cell division, septation initiation signaling cascade, intracellular protein kinase cascade, barrier septum formation, protein phosphorylation, intracellular signal transduction, cytoskeleton organization, cellular response to stimulus, or in RNA, ncRNA and rRNA processing. Importantly, proteins with significantly down-regulated phosphorylation were specifically enriched for R-X-X-S and R-X-R-X-X-S motifs, which are typical consensus substrate sequences for phosphorylation by the AGC family of kinases. The results of this study provide a basis for further analysis of the role of the Ksg1 kinase and its targets in S. pombe and may also be useful for studying Ksg1 orthologs in other organisms.

Phosphoproteomic analysis reveals CDK5-Mediated phosphorylation of MTDH inhibits protein synthesis in microglia

CDK5 plays a crucial role in maintaining normal central nervous system (CNS) development and synaptic function, while microglia are the primary immune cells present in the CNS and play vital physiological roles in CNS development, immune surveillance, and regulation of synaptic plasticity. Despite this, our understanding of both the substrate proteins and functional mechanisms of CDK5 in microglia remains limited. To address this, we utilized CRISPR-Cas9 knockout of Cdk5 in BV2 cells and conducted quantitative phosphoproteomics analysis to systematically screen potential CDK5 substrates in microglia. Our findings identified 335 phosphorylation sites on 234 proteins as potential CDK5 substrates in microglia based on the reported sequence motif. Through in vitro kinase assay and intracellular inhibition and knockout of CDK5 experiments, we confirmed that ER proteins MTDH (protein LYRIC) and Calnexin are novel substrate proteins of CDK5. Moreover, we demonstrated for the first time a critical mechanism for regulating protein synthesis in microglia, that the phosphorylation of S565 site on MTDH, a key protein mediating cell growth, by CDK5 inhibits protein synthesis. Our data provide valuable insights for the discovery of new substrate proteins of CDK5 and the in-depth investigation of the function and mechanism of CDK5 in microglia.

PTMoreR-enabled cross-species PTM mapping and comparative phosphoproteomics across mammals

To support PTM proteomic analysis and annotation in different species, we developed PTMoreR, a user-friendly tool that considers the surrounding amino acid sequences of PTM sites during BLAST, enabling a motif-centric analysis across species. By controlling sequence window similarity, PTMoreR can map phosphoproteomic results between any two species, perform site-level functional enrichment analysis, and generate kinase-substrate networks. We demonstrate that the majority of real P-sites in mice can be inferred from experimentally derived human P-sites with PTMoreR mapping. Furthermore, the compositions of 129 mammalian phosphoproteomes can also be predicted using PTMoreR. The method also identifies cross-species phosphorylation events that occur on proteins with an increased tendency to respond to the environmental factors. Moreover, the classic kinase motifs can be extracted across mammalian species, offering an evolutionary angle for refining current motifs. PTMoreR supports PTM proteomics in non-human species and facilitates quantitative phosphoproteomic analysis.

Comparative quantitative phosphoproteomic and parallel reaction monitoring analysis of soybean roots under aluminum stress identify candidate phosphoproteins involved in aluminum resistance capacity

Aluminum (Al) toxicity adversely impacts soybean (Glycine max) growth in acidic soil. Reversible protein phosphorylation plays an important role in adapting to adverse environmental conditions by regulating multiple physiological processes including signal transduction, energy coupling and metabolism adjustment in higher plant. This study aimed to reveal the Al-responsive phosphoproteins to understand their putative function and involvement in the regulation of Al resistance in soybean root. We used immobilized metal affinity chromatography to enrich the key phosphoproteins from soybean root apices at 0, 4, or 24 h Al exposure. These phosphoproteins were detected using liquid chromatography-tandem mass spectrometry measurement, verified by parallel reaction monitoring (PRM), and functionally characterized via overexpression in soybean hairy roots. A total of 638 and 686 phosphoproteins were identified as differentially enriched between the 4-h and 0-h, and the 24-h and 0-h Al treatment comparison groups, respectively. Typically, the phosphoproteins involved in biological processes including cell wall modification, and RNA and protein metabolic regulation displayed patterns of decreasing enrichment (clusters 3, 5 and 6), however, the phosphoproteins involved in the transport and metabolic processes of various substrates, and signal transduction pathways showed increased enrichment after 24 h of Al treatment. The enrichment of phosphoproteins in organelle organization bottomed after 4 h of Al treatment (cluster 1). Next, we selected 26 phosphoproteins from the phosphoproteomic profiles, assessed their enrichment status using PRM, and detected enrichment patterns similar to those observed via phosphoproteomic analysis. Among them, 15 phosphoproteins were found to reduce the accumulation of Al and callose in Al-stressed soybean root apices when their corresponding genes were individually overexpressed in soybean hairy roots. In summary, the findings of this study facilitated a comprehensive understanding of the protein phosphorylation events involved in Al resistance responses and revealed some critical phosphoproteins that enhance Al resistance in soybean roots.

In-depth phosphoproteomic profiling of the insulin signaling response in heart tissue and cardiomyocytes unveils canonical and specialized regulation

BackgroundInsulin signaling regulates cardiac substrate utilization and is implicated in physiological adaptations of the heart. Alterations in the signaling response within the heart are believed to contribute to pathological conditions such as type-2 diabetes and heart failure. While extensively investigated in several metabolic organs using phosphoproteomic strategies, the signaling response elicited in cardiac tissue in general, and specifically in the specialized cardiomyocytes, has not yet been investigated to the same extent.MethodsInsulin or vehicle was administered to male C57BL6/JRj mice via intravenous injection into the vena cava. Ventricular tissue was extracted and subjected to quantitative phosphoproteomics analysis to evaluate the insulin signaling response. To delineate the cardiomyocyte-specific response and investigate the role of Tbc1d4 in insulin signal transduction, cardiomyocytes from the hearts of cardiac and skeletal muscle-specific Tbc1d4 knockout mice, as well as from wildtype littermates, were studied. The phosphoproteomic studies involved isobaric peptide labeling with Tandem Mass Tags (TMT), enrichment for phosphorylated peptides, fractionation via micro-flow reversed-phase liquid chromatography, and high-resolution mass spectrometry measurements.ResultsWe quantified 10,399 phosphorylated peptides from ventricular tissue and 12,739 from isolated cardiomyocytes, localizing to 3,232 and 3,128 unique proteins, respectively. In cardiac tissue, we identified 84 insulin-regulated phosphorylation events, including sites on the Insulin Receptor (InsrY1351, Y1175, Y1179, Y1180) itself as well as the Insulin receptor substrate protein 1 (Irs1S522, S526). Predicted kinases with increased activity in response to insulin stimulation included Rps6kb1, Akt1 and Mtor. Tbc1d4 emerged as a major phosphorylation target in cardiomyocytes. Despite limited impact on the global phosphorylation landscape, Tbc1d4 deficiency in cardiomyocytes attenuated insulin-induced Glut4 translocation and induced protein remodeling. We observed 15 proteins significantly regulated upon knockout of Tbc1d4. While Glut4 exhibited decreased protein abundance consequent to Tbc1d4-deficiency, Txnip levels were notably increased. Stimulation of wildtype cardiomyocytes with insulin led to the regulation of 262 significant phosphorylation events, predicted to be regulated by kinases such as Akt1, Mtor, Akt2, and Insr. In cardiomyocytes, the canonical insulin signaling response is elicited in addition to regulation on specialized cardiomyocyte proteins, such as Kcnj11Y12 and DspS2597. Details of all phosphorylation sites are provided.ConclusionWe present a first global outline of the insulin-induced phosphorylation signaling response in heart tissue and in isolated adult cardiomyocytes, detailing the specific residues with changed phosphorylation abundances. Our study marks an important step towards understanding the role of insulin signaling in cardiac diseases linked to insulin resistance.Graphical

Quantitative Phosphoproteomic Profiling of Mouse Sperm Maturation in Epididymis Revealed Kinases Important for Sperm Motility

Transcriptionally and translationally silent sperm undergo functional maturation during epididymis traverse, which provides sperm ability to move and is crucial for successful fertilization. However, the molecular mechanisms governing sperm maturation remain poorly understood, especially at the protein post-translational modification level. In this study, we conducted a comprehensive quantitative phosphoproteomic analysis of mouse epididymal sperm from different regions (caput, corpus, and cauda) to unveil the dynamics of protein phosphorylation during sperm maturation. We identified 6447 phosphorylation sites in 1407 phosphoproteins, and 345 phosphoproteins were differentially phosphorylated between caput and cauda sperm. Gene ontology and KEGG pathway analyses showed enrichment of differentially phosphorylated proteins in energy metabolism, sperm motility, and fertilization. Kinase substrate network analysis followed by inhibition assay and quantitative phosphoproteomics analysis showed that TSSK2 kinase is important for sperm motility and progressive motility. This study systemically characterized the intricate phosphorylation regulation during sperm maturation in the mouse epididymis, which can be a basis to elucidate sperm motility acquisition, and to offer potential targets for male contraception and the treatment of male infertility.

Defense response to caterpillar feeding stress in wild Pinus tabuliformis unveiled by quantitative integrated proteomic and phosphoproteomic analyses

Defense response to caterpillar feeding stress in wild Pinus tabuliformis unveiled by quantitative integrated proteomic and phosphoproteomic analyses

Deciphering the phospho-signature induced by hepatitis B virus in primary human hepatocytes.

Phosphorylation is a major post-translation modification (PTM) of proteins which is finely tuned by the activity of several hundred kinases and phosphatases. It controls most if not all cellular pathways including anti-viral responses. Accordingly, viruses often induce important changes in the phosphorylation of host factors that can either promote or counteract viral replication. Among more than 500 kinases constituting the human kinome only few have been described as important for the hepatitis B virus (HBV) infectious cycle, and most of them intervene during early or late infectious steps by phosphorylating the viral Core (HBc) protein. In addition, little is known on the consequences of HBV infection on the activity of cellular kinases. The objective of this study was to investigate the global impact of HBV infection on the cellular phosphorylation landscape early after infection. For this, primary human hepatocytes (PHHs) were challenged or not with HBV, and a mass spectrometry (MS)-based quantitative phosphoproteomic analysis was conducted 2- and 7-days post-infection. The results indicated that while, as expected, HBV infection only minimally modified the cell proteome, significant changes were observed in the phosphorylation state of several host proteins at both time points. Gene enrichment and ontology analyses of up- and down-phosphorylated proteins revealed common and distinct signatures induced by infection. In particular, HBV infection resulted in up-phosphorylation of proteins involved in DNA damage signaling and repair, RNA metabolism, in particular splicing, and cytoplasmic cell-signaling. Down-phosphorylated proteins were mostly involved in cell signaling and communication. Validation studies carried out on selected up-phosphorylated proteins, revealed that HBV infection induced a DNA damage response characterized by the appearance of 53BP1 foci, the inactivation of which by siRNA increased cccDNA levels. In addition, among up-phosphorylated RNA binding proteins (RBPs), SRRM2, a major scaffold of nuclear speckles behaved as an antiviral factor. In accordance with these findings, kinase prediction analysis indicated that HBV infection upregulates the activity of major kinases involved in DNA repair. These results strongly suggest that HBV infection triggers an intrinsic anti-viral response involving DNA repair factors and RBPs that contribute to reduce HBV replication in cell culture models.

The cAMP-dependent phosphorylation footprint in response to heat stress

Key messagecAMP modulates the phosphorylation status of highly conserved phosphosites in RNA-binding proteins crucial for mRNA metabolism and reprogramming in response to heat stress.In plants, 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) is a second messenger that modulates multiple cellular targets, thereby participating in plant developmental and adaptive processes. Although its role in ameliorating heat-related damage has been demonstrated, mechanisms that govern cAMP-dependent responses to heat have remained elusive. Here we analyze the role cAMP–dependent phosphorylation during prolonged heat stress (HS) with a view to gain insight into processes that govern plant responses to HS. To do so, we performed quantitative phosphoproteomic analyses in Nicotiana tabacum Bright Yellow-2 cells grown at 27 °C or 35 °C for 3 days overexpressing a molecular “sponge” that reduces free intracellular cAMP levels. Our phosphorylation data and analyses reveal that the presence of cAMP is an essential factor that governs specific protein phosphorylation events that occur during prolonged HS in BY-2 cells. Notably, cAMP modulates HS-dependent phosphorylation of proteins that functions in mRNA processing, transcriptional control, vesicular trafficking, and cell cycle regulation and this is indicative for a systemic role of the messenger. In particular, changes of cAMP levels affect the phosphorylation status of highly conserved phosphosites in 19 RNA-binding proteins that are crucial during the reprogramming of the mRNA metabolism in response to HS. Furthermore, phosphorylation site motifs and molecular docking suggest that some proteins, including kinases and phosphatases, are conceivably able to directly interact with cAMP thus further supporting a regulatory role of cAMP in plant HS responses.

Abstract PO2-16-04: Proteogenomic characterization of non-luminal A versus luminal A breast cancer tumor cells enriched by laser microdissection

Abstract Introduction: Breast cancer (BC) is classified into four widely-accepted intrinsic subtypes based on PAM (Prediction Analysis of Microarray) 50 gene expression profiles: Luminal A (LumA), Luminal B (LumB), Her2-enriched (Her2) and basal-like (Basal). Recent multi-omic studies of human BC have identified many potential therapeutic biomarkers for these subtypes and have also revealed the extensive molecular heterogeneity of the disease. Patients with LumA tumors generally have better outcomes because these tumors are typically slower-growing and responsive to hormone therapy. However, there is still considerable variation in the clinical outcomes of patients with non-LumA tumors. Here, we strive to understand the proteogenomic characteristics of intrinsically-defined non-LumA tumors in reference to LumA tumors. Methods: A total of 117 retrospectively-collected, untreated primary breast tumor specimens, with a focus on non-LumA subtypes, were selected from the Clinical Breast Care Project and consented using a HIPAA-compliant, IRB-approved protocol. The study cohort had a median patient follow-up of 9.3 years, enabling clinical outcome-related analyses. Immunohistochemistry (IHC) subtyping was used to enrich the cohort with non-LumA subtypes. Breast tumors were embedded in OCT (Optimal Cutting Temperature) compound and processed by laser microdissection (LMD) to enrich for tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor using the illustra triplePrep kit. Paired-end RNA sequencing and whole genome sequencing (WGS) were performed for 117 and 99 tumors, respectively, using the Illumina HiSeq platform. Quantitative global proteomics and phosphoproteomic analyses were performed on 112 and 50 tumors, respectively, using isobaric TMT 6-plex labeling with the “universal reference” strategy. Results: We observed significantly lower stromal, immune and microenvironment scores in non-basal-like tumors, especially the LumA subtype from LMD-processed samples, compared to that of the same tumors but bulk-processed in TCGA-breast cancer study. There was also significantly lower stromal gene expression in LMD LumA compared to TCGA LumA. Unlike a recent report on proteomics clustering of bulk-processed tumors, we did not observe a stromal-enriched cluster, probably because the use of LMD minimized stromal components. Many common patterns of somatic mutations were observed among non-LumA tumors, such as dominant TP53 mutations and 5q deletion, suggesting a potentially common cell-of-origin for non-LumA tumors, such as an ER negative cancer stem cell or progenitor cell for these tumor subtypes. Cell proliferation-associated genes were amplified and proliferation pathways strongly enriched in non-LumA tumors. In addition to this, we identified two distinct phosphoproteomic profiles for relapsed and relapse-free basal-like BC. Moreover, we also identified 17 differentially expressed phosphopeptides that could identify cases of relapsed and relapse-free basal-like BC with a significant difference in progression-free interval of surviving cases. Conclusion: This study provides an integrated proteogenomic characterization of non-LumA vs LumA tumor specimens enriched for cancer cells. We identified many common features of non-LumA tumors and also identified phosphopeptides that could serve as potential biomarkers for less aggressive basal-like BC and possibly guide treatment selections. Disclaimer: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of USUHS, HJF, the DoD or the Departments of the Army, Navy or Air Force or the DOE or PNNL. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Praveen Kumar Raj Kumar, Xiaoying Lin, Tao Liu, Lori Sturtz, Marina Gritsenko, Vladislav Petyuk, Tyler Sagendorf, Brenda Deyarmin, Jianfang Liu, Anupama Praveen-Kumar, Guisong Wang, Jason McDermott, Anil Shukla, Ronald Moore, Matthew Monroe, Bobbie-Jo Webb-Robertson, Jeffrey Hooke, Leigh Fantacone-Campbell, Brad Mostoller, Leonid Kvecher, Jennifer Kane, Jennifer Melley, Stella Somiari, Patrick Soon-Shiong, Richard Smith, Richard Mural, Karin Rodland, Craig Shriver, Albert Kovatich, Hai Hu. Proteogenomic characterization of non-luminal A versus luminal A breast cancer tumor cells enriched by laser microdissection [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-16-04.

Upregulated Matrisomal Proteins and Extracellular Matrix Mechanosignaling Underlie Obesity-Associated Promotion of Pancreatic Ductal Adenocarcinoma.

Diet-induced obesity (DIO) promotes pancreatic ductal adenocarcinoma (PDAC) in mice expressing KRasG12D in the pancreas (KC mice), but the precise mechanisms remain unclear. Here, we performed multiplex quantitative proteomic and phosphoproteomic analysis by liquid chromatography-tandem mass spectrometry and further bioinformatic and spatial analysis of pancreas tissues from control-fed versus DIO KC mice after 3, 6, and 9 months. Normal pancreatic parenchyma and associated proteins were steadily eliminated and the novel proteins, phosphoproteins, and signaling pathways associated with PDAC tumorigenesis increased until 6 months, when most males exhibited cancer, but females did not. Differentially expressed proteins and phosphoproteins induced by DIO revealed the crucial functional role of matrisomal proteins, which implies the roles of upstream regulation by TGFβ, extracellular matrix-receptor signaling to downstream PI3K-Akt-mTOR-, MAPK-, and Yap/Taz activation, and crucial effects in the tumor microenvironment such as metabolic alterations and signaling crosstalk between immune cells, cancer-associated fibroblasts (CAFs), and tumor cells. Staining tissues from KC mice localized the expression of several prognostic PDAC biomarkers and elucidated tumorigenic features, such as robust macrophage infiltration, acinar-ductal metaplasia, mucinous PanIN, distinct nonmucinous atypical flat lesions (AFLs) surrounded by smooth muscle actin-positive CAFs, invasive tumors with epithelial-mesenchymal transition arising close to AFLs, and expanding deserted areas by 9 months. We next used Nanostring GeoMX to characterize the early spatial distribution of specific immune cell subtypes in distinct normal, stromal, and PanIN areas. Taken together, these data richly contextualize DIO promotion of Kras-driven PDAC tumorigenesis and provide many novel insights into the signaling pathways and processes involved.

Quantitative Phosphoproteomics Analysis Reveals the Protective Mechanism of Chlorogenic Acid on Immunologically Stressed Broiler Meat Quality.

Modern poultry production is stressful for the birds, and this stress is recognized as a major cause of inferior meat quality. Chlorogenic acid (CGA), a plant phenolic acid, has excellent antioxidant and anti-inflammatory properties. The antioxidant capacity and phosphoproteomics of immunologically stressed broiler breast muscle were assessed to elucidate the mechanism of the beneficial effects of CGA on meat quality. Dietary CGA decreased drip and cooking loss, postmortem pH and antioxidant capacity of breast muscle from stressed broilers, and increased MyHC-I mRNA levels. Quantitative phosphoproteomics revealed that CGA supplementation downregulated the phosphorylation of myofibrillar proteins, glycolytic enzymes, and endoplasmic reticulum proteins involved in homeostasis, which contributed to improving the meat quality of broilers. Moreover, 14 phosphorylation sites (e.g., P13538-Ser1236 and F1NN63-Ser117) in 13 phosphoproteins were identified as key regulators of processes related to broiler meat quality. Together, these findings provide novel regulatory targets and nutritional strategies for improving the stressed broiler meat quality.

Surviving winter on the Qinghai-Xizang Plateau: Extensive reversible protein phosphorylation plays a dominant role in regulating hypometabolism in hibernating Nanorana parkeri.

Changes in protein abundance and reversible protein phosphorylation (RPP) play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes. To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation, we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog, Nanorana parkeri, living on the Qinghai-Xizang (Tibet) Plateau (QTP). In total, 5 170 proteins and 5 695 phosphorylation sites in 1 938 proteins were quantified. Based on proteomic analysis, 674 differentially expressed proteins (438 up-regulated, 236 down-regulated) were screened in hibernating N. parkeri versus summer individuals. Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways, whereas lower expressed proteins were mainly involved in metabolic processes. A total of 4 251 modified sites (4 147 up-regulated, 104 down-regulated) belonging to 1 638 phosphoproteins (1 555 up-regulated, 83 down-regulated) were significantly changed in the liver. During hibernation, RPP regulated a diverse array of proteins involved in multiple functions, including metabolic enzymatic activity, ion transport, protein turnover, signal transduction, and alternative splicing. These changes contribute to enhancing protection, suppressing energy-consuming processes, and inducing metabolic depression. Moreover, the activities of phosphofructokinase, glutamate dehydrogenase, and ATPase were all significantly lower in winter compared to summer. In conclusion, our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.

Quantitative phosphoproteomic analysis of chicken DF-1 cells infected with Eimeria tenella, using tandem mass tag (TMT) and parallel reaction monitoring (PRM) mass spectrometry.

Eimeria tenella is an obligate intracellular parasite which causes great harm to the poultry breeding industry. Protein phosphorylation plays a vital role in host cell-E. tenella interactions. However, no comprehensive phosphoproteomic analyses of host cells at various phases of E. tenella infection have been published. In this study, quantitative phosphoproteomic analysis of chicken embryo DF-1 fibroblasts that were uninfected (UI) or infected with E. tenella for 6h (PI6, the early invasion phase) or 36h (PI36, the trophozoite development phase) was conducted. A total of 10,122 phosphopeptides matched to 3,398 host cell phosphoproteins were identified and 13,437 phosphorylation sites were identified. Of these, 491, 1,253, and 275 differentially expressed phosphorylated proteins were identified in the PI6/UI, PI36/UI, and PI36/PI6 comparisons, respectively. KEGG pathway enrichment analysis showed that E. tenella modulated host cell processes through phosphorylation, including focal adhesion, regulation of the actin cytoskeleton, and FoxO signaling to support its early invasion phase, and modulating adherens junctions and the ErbB signaling pathway to favor its trophozoite development. These results enrich the data on the interaction between E. tenella and host cells and facilitate a better understanding of the molecular mechanisms underlying host-parasite relationships.

Using CRISPR-Cas9/phosphoproteomics to identify substrates of calcium/calmodulin-dependent kinase 2δ

Ca2+/Calmodulin-dependent protein kinase 2 (CAMK2) family proteins are involved in the regulation of cellular processes in a variety of tissues including brain, heart, liver, and kidney. One member, CAMK2δ (CAMK2D), has been proposed to be involved in vasopressin signaling in the renal collecting duct, which controls water excretion through regulation of the water channel aquaporin-2 (AQP2). To identify CAMK2D target proteins in renal collecting duct cells (mpkCCD), we deleted Camk2d and carried out LC-MS/MS-based quantitative phosphoproteomics. Specifically, we used CRISPR/Cas9 with two different guide RNAs targeting the CAMK2D catalytic domain to create multiple CAMK2D KO cell lines. AQP2 protein abundance was lower in the CAMK2D KO cells than in CAMK2D-intact controls. AQP2 phosphorylation at Ser256 and Ser269 (normalized for total AQP2) was decreased. However, trafficking of AQP2 to and from the apical plasma membrane was sustained. Large-scale quantitative phosphoproteomic analysis (TMT-labeling) in the presence of the vasopressin analog dDAVP (0.1nM, 30min) allowed quantification of 11,570 phosphosites of which 169 were significantly decreased, while 206 were increased in abundance in CAMK2D KO clones. These data are available for browsing or download at https://esbl.nhlbi.nih.gov/Databases/CAMK2D-proteome/. Motif analysis of the decreased phosphorylation sites revealed a target preference of -(R/K)-X-X-p(S/T)-X-(D/E), matching the motif identified in previous invitro phosphorylation studies using recombinant CAMK2D. Thirty five of the significantly downregulated phosphorylation sites in CAMK2D KO cells had exactly this motif and are judged to be likely direct CAMK2D targets. This adds to the list of known CAMK2D target proteins found in prior reductionist studies.

Quantitative Phosphoproteomic Analysis Provides Insights into the Sodium Bicarbonate Responsiveness of Glycine max.

Sodium bicarbonate stress caused by NaHCO3 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 NaHCO3 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.

PB0390 Identification of Key Regulators of Procoagulant COAT Platelet Generation by Quantitative Phosphoproteomic Analysis and Phosphoflow

PB0390 Identification of Key Regulators of Procoagulant COAT Platelet Generation by Quantitative Phosphoproteomic Analysis and Phosphoflow

Quantitative Proteomic and Phosphoproteomic Analyses Reveal a Role of Death-Associated Protein Kinase 1 in Regulating Hippocampal Synapse.

Death-associated protein kinase 1 (DAPK1) is a stress-responsive calcium/calmodulin (CaM)-regulated serine/threonine protein kinase that is actively involved in stress-induced cell death. The dysregulation of DAPK1 has been established in various neurological disorders such as epilepsy, Alzheimer's disease (AD), and Parkinson's disease (PD). Recent research indicates a synaptic localization of DAPK1 in neurons, suggesting a potential role of DAPK1 in modulating synaptic structure and function. However, the key molecules and pathways underlying the influence of DAPK1 on synapses remain elusive. We utilized quantitative proteomic and phosphoproteomic analyses to compare the differences in protein expression and phosphorylation in hippocampal tissues of wild-type (WT) and DAPK1-knockout (KO) mice. Bioinformatic analysis of differentially expressed proteins and phosphoproteins revealed a preferential enrichment of proteins involved in regulating synaptic function, cytoskeletal structure, and neurotransmission. Gene set enrichment analysis (GESA) highlighted altered presynaptic functions including synaptic vesicle priming and glutamate secretion in KO mice. Besides, we observed that proteins with potential phosphorylation motifs of ERK and DAPK1 were overrepresented among the differential phosphoproteins and were highly enriched in neuronal function-related pathways. Furthermore, Western blot analysis validated differences in the expression of several proteins closely associated with presynaptic organization, dendrites and calcium transmembrane transport between KO and WT mice, further corroborating the potential involvement of DAPK1 in the regulation of synaptic functions. Overall, our data provide molecular evidence to elucidate the physiological links between DAPK1 and neuronal functions and help clarify the role of DAPK1 in the pathogenesis of neurodevelopmental and neurodegenerative diseases.

Quantitative proteomic and phosphoproteomic analysis reveal the relationship between mitochondrial dysfunction and cytoskeletal remodeling in hiPSC-CMs deficient in PINK1

BackgroundHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are seed cells that can be used for alternative treatment of myocardial damage. However, their immaturity limits their clinical application. Mitochondrial development accompanies cardiomyocyte maturation, and PINK1 plays an important role in the regulation of mitochondrial quality. However, the role and mechanism of PINK1 in cardiomyocyte development remain unclear.MethodsWe used proteomic and phosphoproteomic to identify protein and phosphosite changes in hiPSC-CMs deficient in PINK1. Bioinformatics analysis was performed to identify the potential biological functions and regulatory mechanisms of these differentially expressed proteins and validate potential downstream mechanisms.ResultsDeletion of PINK1 resulted in mitochondrial structural breakdown and dysfunction, accompanied by disordered myofibrils arrangement. hiPSC-CMs deficient in PINK1 exhibited significantly decreased expression of mitochondrial ATP synthesis proteins and inhibition of the oxidative phosphorylation pathway. In contrast, the expression of proteins related to cardiac pathology was increased, and the phosphoproteins involved in cytoskeleton construction were significantly altered. Mechanistically, PINK1 deletion damaged the mitochondrial cristae of hiPSC-CMs and reduced the efficiency of mitochondrial respiratory chain assembly.ConclusionThe significantly differentially expressed proteins identified in this study highlight the important role of PINK1 in regulating mitochondrial quality in hiPSC-CMs. PINK1-mediated mitochondrial respiratory chain assembly is the basis for mitochondrial function. Whereas the cytoskeleton may be adaptively altered in response to mitochondrial dysfunction caused by PINK1 deletion, inadequate energy supply hinders myocardial development. These findings facilitate the exploration of the mechanism of PINK1 in cardiomyocyte development and guide efforts to promote the maturation of hiPSC-CMs.