Phosphoproteomic Studies Research Articles

An eco-friendly, low-cost, and automated strategy for phosphoproteome profiling

An automated, online analysis platform using a reusable phos-trap column helps reduce organic solvent, plastic consumables, waste, and labor costs in phosphoproteomic studies.

Superhydrophilic nanocomposite adsorbents modified via nitrogen-rich phosphonate-functionalized ionic liquid linkers: enhanced phosphopeptide enrichment and phosphoproteome analysis of tau phosphorylation in the hippocampal lysate of Alzheimer's transgenic mice.

In this study, new graphene-based IMAC nanocomposites for phosphopeptide enrichment were prepared according to the guideline of our new design strategy. Superhydrophilic polyethyleneimine (PEI) was introduced, to which a phosphonate-functionalized ionic liquid (PFIL) was covalently bound, to form superhydrophilic and cationic surface layers with high densities of nitrogen atoms, phosphonate functional groups, and high-loading metal ions. Due to the combined features of superhydrophilicity, flexibility, highly dense metal binding sites, large surface area and excellent size-exclusion effect, the fabricated nanocomposite G@mSiO2@PEI-PFIL-Ti4+ exhibits superior detection sensitivity to enrich phosphopeptides (tryptic β-casein digest, 0.1 fmol), and extraordinary enrichment specificity to enrich phosphopeptides from a digest mixture of β-casein and bovine serum albumin (BSA) (molar ratio, 1 : 12 000). The excellent size-exclusion effect was also observed, and 27 endogenous phosphopeptides were identified in human saliva. All these results could be attributed to the unique superhydrophilic nanocomposite structure with a high density of a cationic linker modified with phosphonate functionality. Moreover, G@mSiO2@PEI-PFIL-Ti4+ adsorbents were used to extract phosphopeptides from the tryptic digests of hippocampal lysates for quantitative phosphoproteome analysis. The preliminary results indicate that 1649 phosphoproteins, 3286 phosphopeptides and 4075 phosphorylation sites were identified. A total of 13 Alzheimer's disease (AD)-related phosphopeptides within tau proteins were detected with a wide coverage from p-Thr111 to p-Ser404, in which the amounts of some phoshopeptides at certain sites in AD transgenic mice were found statistically higher than those in wild type littermates. Besides, phosphorylated neurofilament heavy chains, a potential biomarker for amyotrophic lateral sclerosis and traumatic brain injury, were also identified. Finally, the adsorbent was applied to human cerebrospinal fluid (CSF) and blood samples. 5 unique phosphopeptides of neuroendocrine specific VGF were identified in the CSF, while many phosphopeptides originated from the nervous system were found in the blood sample. All these results suggest that our new IMAC materials exhibit unbiased enrichment ability with superior detection sensitivity and specificity, allowing the global phosphoproteome analysis of complicated biological samples more convincible and indicating the potential use in disease diagnosis.

Plant Phosphoproteomics: Known Knowns, Known Unknowns, and Unknown Unknowns of an Emerging Systems Science Frontier.

Plant systems science research depends on the dynamic functional maps of the biological substrates of plant phenotypes and host/environment interactions in diverse ecologies. In this context, high-resolution mass spectrometry platforms offer comprehensive insights into the molecular pathways regulated by protein phosphorylation. Reversible protein phosphorylation is a ubiquitous reaction in signal transduction mechanisms in biological systems. In contrast to human and animal biology research, a plethora of experimental options for functional mapping and regulation of plant biology are, however, not currently available. Plant phosphoproteomics is an emerging field of research that aims at addressing this gap in systems science and plant omics, and thus has a large scope to empower fundamental discoveries. To date, large-scale data-intensive identification of phosphorylation events in plants remained technically challenging. In this expert review, we present a critical analysis and overview of phosphoproteomic studies performed in the model plant Arabidopsis thaliana. We discuss the technical strategies used for the enrichment of phosphopeptides and methods used for their quantitative assessment. Various types of mass spectrometry data acquisition and fragmentation methods are also discussed. The insights gathered here can allow plant biology and systems science researchers to design high-throughput function-oriented experimental workflows that elucidate the regulatory signaling mechanisms impacting plant physiology and plant diseases.

A Decade of Pollen Phosphoproteomics.

Angiosperm mature pollen represents a quiescent stage with a desiccated cytoplasm surrounded by a tough cell wall, which is resistant to the suboptimal environmental conditions and carries the genetic information in an intact stage to the female gametophyte. Post pollination, pollen grains are rehydrated, activated, and a rapid pollen tube growth starts, which is accompanied by a notable metabolic activity, synthesis of novel proteins, and a mutual communication with female reproductive tissues. Several angiosperm species (Arabidopsis thaliana, tobacco, maize, and kiwifruit) were subjected to phosphoproteomic studies of their male gametophyte developmental stages, mostly mature pollen grains. The aim of this review is to compare the available phosphoproteomic studies and to highlight the common phosphoproteins and regulatory trends in the studied species. Moreover, the pollen phosphoproteome was compared with root hair phosphoproteome to pinpoint the common proteins taking part in their tip growth, which share the same cellular mechanisms.

Structural Basis of Feedback Control of Oncogenic Signaling in B-Lymphoid Malignancies

Introduction: Multiple structural elements are known that recruit kinases to sites of active signal transduction (e.g. lipid rafts) within the plasma membrane. For instance, pleckstrin homology (PH) domains and the conserved intracellular loop (CIL) motifs recently discovered by us (Lee et al., Nature 2021) direct kinase molecules (Src, BTK, AKT and PI3K) to large signaling clusters in lipid rafts to amplify normal or oncogenic B-cell signaling. While concepts of site-specific recruitment of kinases to initiate signaling are well established, little is known about spatial control of inhibitory phosphatases and how they are directed to sites of maximal signaling strength.Results: Here we discovered the cytoplasmic tail of CD25 as an essential structural element for site-specific recruitment of inhibitory phosphatases. CD25-mediated shuttling of inhibitory phosphatases balances oncogenic signaling strength and is essential for cell-survival in B-cell malignancies, including B-ALL, CLL and mantle cell lymphoma. This was unexpected because CD25 (IL2RA) is known as one of three chains of the Interleukin-2 (IL2) receptor on T- and NK-cells. Studying clinical outcome and gene expression in multiple clinical cohorts, we found CD25 as a top-ranking predictor of poor clinical outcome in patients with B-ALL, mantle cell lymphoma and CLL.Our mechanistic experiments revealed that CD25 expressed on B-cells is monomeric. Unlike T- and NK-cells, CD25 expressed on activated or transformed B-cells does not form a heterotrimeric IL2 receptor with the IL2Rb and common g-chain. Instead of contributing to IL2-signaling, CD25 showed dynamic recruitment either to the B-cell receptor (BCR) or transforming oncogenes within lipid rafts. Conditional deletion of CD25 at earliest stages of B-cell development in mice resulted in profound B-cell defects. However, these defects were not replicated by IL2-deficiency and B-cell development in Il2-/- mice was normal.Inducible deletion of CD25 in genetic models for B-ALL and mantle cell lymphoma revealed an essential function of CD25 in maintaining homeostasis of signaling strength. In the absence of CD25, B-ALL and mantle cell lymphoma cells showed autonomous Ca 2+ oscillations, constitutive activation of Src-family kinases, BTK and ERK as well as the AMPK energy-stress sensor. CD25-deficient B-ALL and mantle cell lymphoma cells initially showed increased proliferation but rapidly died from exhaustion. Consistent with massive accumulation of p53, CD25-deficient B-ALL cells failed to form colonies and to initiate leukemia in transplant-recipient mice.Our mechanistic studies focused on the short (13 residues) cytoplasmic tale of CD25, which features a prominent serine/threonine kinase substrate motif. In vitro kinase assays for 62 candidate serine/threonine kinases revealed PKCd as top-ranking kinase and the serine residue 268 in the tail of CD25 as its principal substrate. The proximity-based labeling assays (BioID) revealed that the CD25 tail recruits PKCd and its adapter RACK1, which scaffold the inhibitory phosphatases SHIP1 and SHP1 for site-specific activation in lipid rafts (see Schematic). Consistent with these results, phospho-proteomic studies revealed that inducible deletion of CD25 resulted in acute inactivation of SHIP1 and SHP1 as well as the inhibitory RACK1 scaffold. Multiscale molecular dynamics (MD) simulations revealed that PKCd-mediated phosphorylation of the CD25-tail (S268) tightened interactions with RACK1 and SHIP1 and SHP1 phosphatases, whereas these interactions were destabilized when the CD25-tail was mutated (S268A).Conclusions: Mechanisms of site-specific recruitment of kinases for signal amplification have been extensively studied. However, structural elements that direct inhibitory phosphatases to sites of maximal signaling activity for feedback control remained largely unknown. Here we discovered a PKCd substrate motif in the cytoplasmic tail of CD25 (S268) as a central element to recruit inhibitory phosphatases, scaffolded by RACK1, to curb excessive signaling activity and energy expenditure. High expression levels of CD25 enable higher baseline activity of oncogenic signaling and are associated with poor patient-outcome. Our preclinical studies based on CD25-antibody-drug-conjugates (ADC) revealed that CD25 as a novel target in refractory B-ALL and mantle cell lymphoma. [Display omitted] DisclosuresWeinstock: SecuraBio: Consultancy; ASELL: Consultancy; Bantam: Consultancy; Abcuro: Research Funding; Verastem: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; AstraZeneca: Consultancy; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.

Quantitative phosphoproteomic analysis reveals chemoresistance-related proteins and signaling pathways induced by rhIL-6 in human osteosarcoma cells

BackgroundIL-6 plays a pivotal role in resistance to chemotherapeutics, including lobaplatin. However, the underlying mechanisms are still unclear. This study was to investigate the changes in phosphoproteins and their related signaling pathways in the process of IL-6-induced chemoresistance to lobaplain in osteosarcoma cells.MethodsWe performed a quantitative phosphoproteomic analysis of the response of SaOS-2 osteosarcoma cells to recombinant human IL-6 (rhIL-6) intervention prior to lobaplatin treatment. The cells were divided into the control group (Con), the lobaplatin group (Lob), and the rhIL-6-and-lobaplatin group (IL-6). Three biological replicates of each group were included. The differentially expressed phosphoproteins were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Netphos 3.1 was used for the prediction of kinases, and STRING was used for the visualization of protein–protein interactions. The conserved motifs surrounding the phosphorylated residues were analyzed using the motif-x algorithm. Western blot analysis was performed to verify the differential expression of p-FLNC, its predicted kinase and the related signaling pathway. The results of the bioinformatic analysis were validated by immunohistochemical staining of clinical specimens.ResultsIn total, 3373 proteins and 12,183 peptides, including 3232 phosphorylated proteins and 11,358 phosphorylated peptides, were identified and quantified. Twenty-three significantly differentially expressed phosphoproteins were identified in the comparison between the IL-6 and Lob groups, and p-FLNC ranked second among these phosphoproteins. GO and KEGG analyses revealed the pivotal role of mitogen-activated protein kinase signaling in drug resistance induced by rhIL-6. Four motifs, namely, -SPxxK-, -RxxSP-, -SP-, and -SPK-, demonstrated higher expression in the IL-6 group than in the Lob group. The western blot analysis results verified the higher expression of p-FLNC, AKT1, and p-ERK and the lower expression of p-JNK in the IL-6 group than in the Con and Lob groups. The immunohistochemical staining results showed that p-FLNC, AKT1 and p-ERK1/2 were highly expressed in platinum-resistant clinical specimens but weakly expressed in platinum-sensitive specimens, and platinum-resistant osteosarcoma specimens demonstrated weak expression of p-JNK.ConclusionsThis phosphoproteomic study is the first to reveal the signature associated with rhIL-6 intervention before lobaplatin treatment in human osteosarcoma cells. p-FLNC, AKT1, and MAPK signaling contributes to resistance to lobaplatin in osteosarcoma SaOS-2 cells and may represent molecular targets to overcome osteosarcoma chemoresistance.

Proteomic and phosphoproteomic analyses identify liver-related signaling in retinal pigment epithelial cells during EMT.

Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is associated with several blinding retinal diseases. Using proteomics and phosphoproteomics studies of human induced pluripotent stem cell-derived RPE monolayers with induced EMT, we capture kinase/phosphatase signaling cascades 1h and 12h after induction to better understand the pathways mediating RPE EMT. Induction by co-treatment with transforming growth factor β and tumor necrosis factor alpha (TGNF) or enzymatic dissociation perturbs signaling in many of the same pathways, with striking similarity in the respective phosphoproteomes at 1 h. Liver hyperplasia and hepatocyte growth factor (HGF)-MET signaling exhibit the highest overall enrichment. We also observe that HGF and epidermal growth factor signaling, two cooperative pathways inhibited by EMT induction, regulate the RPE transcriptional profile.

Abstract MP216: Identification Of Novel Phosphoprotein Signaling Pathways In Human Dilated Cardiomyopathy By Integrative Proteomic And Phosphoproteomic Analysis

Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure, yet the majority of the underlying signaling mechanisms remain poorly characterized. Protein phosphorylation is a key regulatory element with profound effects on the activity and function of signaling networks; however, there is a lack of comprehensive phosphoproteomic studies in human DCM patients. We assessed the hypothesis that an integrative phosphoproteomics analysis of human DCM would reveal novel phosphoprotein candidates involved in disease pathophysiology. Combined proteomic and phosphoproteomic analysis of explanted left ventricular tissue samples from DCM patients ( n =4) and non-failing controls ( n =4) identified 5,570 unique proteins with 13,624 corresponding phosphorylation sites. From these analyses, we identified αT-catenin as a unique candidate protein with a cluster of 4 significantly hyperphosphorylated sites in DCM hearts ( P <0.0001), with no change in total αT-catenin expression at the protein level. Bioinformatics analyses of human datasets and confocal imaging of human and mouse cardiac tissue show highly cardiac-enriched expression of αT-catenin, localized to the cardiomyocyte intercalated disc. High resolution 3-dimensional reconstruction shows elongated intercalated disc morphology in DCM hearts (10.07±0.76 μm in controls vs. 17.20±1.87 μm in DCM, P <0.05, n =3/group), with significantly increased colocalization of αT-catenin with the intercalated disc membrane protein N-cadherin (Pearson’s coefficient 0.55±0.04 in controls vs. 0.71±0.02 in DCM, P <0.05, n =3/group). To investigate the functional role of cardiac αT-catenin phosphorylation, we overexpressed WT protein vs. non-phosphorylatable forms based on the loci identified in DCM hearts, in adult mouse cardiomyocytes using lentiviral transduction. Confocal imaging revealed significant internalization of the phospho-null form, as compared to the prominent intercalated disc staining of the WT protein (17.78±0.79% of WT vs. 9.25±0.49% of 4A mutant, P <0.0001, n =50 cells/group). Together, these findings suggest a critical role for αT-catenin phosphorylation in maintaining cardiac intercalated disc organization in human DCM.

Phosphoproteomic analysis of duck egg yolk provides novel insights into its characteristics and biofunctions.

Although the importance of phosphorylation in the function of proteins is known, investigation of the protein phosphorylation of duck egg yolk (DEY) is still very limited. This study aimed to conduct a detailed phosphoproteomic study of DEY using immobilized metal affinity chromatography and ultra-high liquid chromatography tandem mass spectrometry. A total of 253 phosphorylation sites assigned to 66 phosphoproteins were identified in DEY, of which VTG-1, VTG-2, and fibrinogen alpha chain were found to be the highly phosphorylated proteins in DEY. The biological functions of the identified phosphoproteins were illuminated through gene ontology analysis, which showed that they were mainly involved in binding, catalytic, immune response, and metabolic activity. S-X-E and S-X-S were found to be the most conserved serine motifs of phosphorylation in DEY. The comparison of DEY phosphoproteins with those of chicken egg yolk (CEY) revealed that differences mostly involved molecular functions and biological processes. The comparison also revealed a higher phosphorylation level in DEY proteins. The higher phosphorylation level in DEY proteins than that in CEY proteins are supposed to help enhance duck growth performance and biological activities (e.g. antibacterial and antioxidant ability) for better adapting the humid environment the duck lived. © 2021 Society of Chemical Industry.

A phosphoproteomics study reveals a defined genetic program for neural lineage commitment of neural stem cells induced by olfactory ensheathing cell-conditioned medium

Since both Olfactory ensheathing cells (OECs) and neural stem cells (NSCs) have shown certain efficacy in the cellular therapy of nerve injury and disease, there have been a series of investigations in recent years looking at the co-culture of NSCs and OECs. Protein phosphorylation forms the basis for identifying a variety of cellular signaling pathways responsible for regulating the self-renewal and differentiation of NSCs induced by OECs. To better understand the signaling cascades in the early phases of OEC-induced NSC differentiation, changes in the NSC proteome and phosphoproteome during the first 24 h were determined using dimethyl labeling and TiO2 phosphorylation enrichment coupled with Liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 565 proteins and 2511 phosphorylation sites were identified. According to quantitative phosphoproteomics analyses of NSC differentiation induced by OECs during the first 12 and 24 h, it was speculated that there were at least two different signal waves: one peaking within 12 h after stimulation and the second upsurge after 24 h. In addition to understanding the dynamics of the proteome and phosphoproteome in the early stages of NSC differentiation, our analyses identified a key role of the TGF-β3 protein secreted by OECs, which may be an initiating factor that promotes differentiation of NSCs into neurons induced by OECs. These findings not only redemonstrated a OECs-based therapeutic strategy in cell therapy, but also added a node to the regulatory network for the neural lineage commitment of NSCs induced by OECs.

Phosphoproteomic response mapping to anti-angiogenic agents bevacizumab and cediranib in high-grade serous ovarian and endometrial cancer cell lines

<h3>Objectives:</h3> Antiangiogenics have become a cornerstone of treatment in advanced stage and recurrent high grade serous ovarian cancer. Both cediranib (ICON6) and bevacizumab (ICON7, GOG218) have demonstrated benefit as a maintenance treatment for ovarian cancer patients in clinical trials. Investigators have also explored their role in advanced stage and recurrent endometrial carcinoma (MITO-END 2, Rose 2017, Aghajanian 2011). There have been no head to head trials in gynecologic cancers comparing cediranib to bevacizumab, although in colorectal carcinoma, phase III data suggests equivalent efficacy (HORIZON III). We hypothesize that these drugs, although both categorized as anti-angiogenics, induce unique cellular responses and sought to explore the varying impact of each agent on cell signaling using established serous cell models. <h3>Methods:</h3> CAOV3 ovarian cancer cells and Hec50 endometrial cancer cells were exposed to 1µM bevacizumab or cediranib for 24 hours. Cell lysates were analyzed in duplicate using an antibody-based phosphoproteomic array of 875 phosphoproteins supplied by Kinexus Bioinformatics (Vancouver, BC). Priority signaling effects were defined for each drug and in each cell line as those with a significant (>75%) change from control with an acceptable sum of error (<75%). <h3>Results:</h3> Bevacizumab yielded 13 total leads, and cediranib yielded 9 in CAOV3 ovarian cancer cells (Table 1). Both agents significantly enhanced signaling events on the following epitopes: ABl, EZH2, HGK (2 sites), and IGF1R, indicating that these may be some of the core cellular effects of anti-angiogenic agents. Interestingly, all common leads induced by both agents in CAOV3 cells represent potential pro-oncogenic effects. In Hec50 cells (Table 1), both cediranib and bevacizumab treatment induced a unique set of phosphoproteins compared to the effects of these agents in CAOV3 cells, yet common findings were the phosphorylation of IGFR1 by bevacizumab and the activation of alternative MAP kinases in both cell lines. <h3>Conclusions:</h3> Angiogenesis plays a crucial role in tumor development and metastasis. Blocking angiogenesis using monoclonal antibodies such as bevacizumab or tyrosine kinase inhibitors such as cediranib is a strategy that is proving to be helpful in the care of patients with gynecologic malignancies. These molecular agents have both on-target, therapeutic effects but also may induce pathways of resistance. In these comprehensive phosphoproteomic mapping studies, we noted the activation of signaling pathways which were cell and drug specific. One consistent finding was the enhanced phosphorylation of IGFR1 and alternative MAP kinases that may portend eventual drug resistance.

Progesterone triggers Rho kinase-cofilin axis during in vitro and in vivo endometrial decidualization.

Can a combination of the focussed protein kinase assays and a wide-scale proteomic screen pinpoint novel, clinically relevant players in decidualization in vitro and in vivo? Rho-dependent protein kinase (ROCK) activity is elevated in response to the combined treatment with progesterone and 8-Br-cAMP during in vitro decidualization, mirrored by increase of ROCK2 mRNA and protein levels and the phosphorylation levels of its downstream target Cofilin-1 (CFL1) in secretory versus proliferative endometrium. Decidualization is associated with extensive changes in gene expression profile, proliferation, metabolism and morphology of endometrium, yet only a few underlying molecular pathways have been systematically explored. In vitro decidualization of endometrial stromal cells (ESCs) can be reportedly induced using multiple protocols with variable physiological relevance. In our previous studies, cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA)/prolactin axis that is classically upregulated during decidualization showed dampened activation in ESCs isolated from polycystic ovary syndrome (PCOS) patients as compared to controls. In vitro decidualization studies were carried out in passage 2 ESCs isolated from controls (N = 15) and PCOS patients (N = 9). In parallel, lysates of non-cultured ESCs isolated from proliferative (N = 4) or secretory (N = 4) endometrial tissue were explored. The observed trends were confirmed using cryo-cut samples of proliferative (N = 3) or secretory endometrium (N = 3), and in proliferative or secretory full tissue samples from controls (N = 8 and N = 9, respectively) or PCOS patients (N = 10 for both phases). The activities of four target kinases were explored using kinase-responsive probes and selective inhibitors in lysates of in vitro decidualized ESCs and non-cultured ESCs isolated from tissue at different phases of the menstrual cycle. In the latter lysates, wide-scale proteomic and phosphoproteomic studies were further carried out. ROCK2 mRNA expression was explored in full tissue samples from controls or PCOS patients. The immunofluorescent staining of phosphorylated CFL1 was performed in full endometrial tissue samples, and in the in vitro decidualized fixed ESCs from controls or PCOS patients. Finally, the cellular migration properties were explored in live in vitro decidualized ESCs. During in vitro decidualization, the activities of PKA, protein kinase B (Akt/PKB), and ROCK are increased while the activity of casein kinase 2 (CK2) is decreased; these initial trends are observable after 4-day treatment (P < 0.05) and are further augmented following the 9-day treatment (P < 0.001) with mixtures containing progesterone and 8-Br-cAMP or forskolin. The presence of progesterone is necessary for activation of ROCK, yet it is dispensable in the case of PKA and Akt/PKB; in comparison to controls, PCOS patient-derived ESCs feature dampened response to progesterone. In non-cultured ESCs isolated from secretory vs proliferative phase tissue, only activity of ROCK is increased (P < 0.01). ROCK2 protein levels are slightly elevated in secretory versus proliferative ESCs (relative mean standard deviation < 50%), and ROCK2 mRNA is elevated in mid-secretory versus proliferative full tissue samples (P < 0.05) obtained from controls but not PCOS patients. Activation of ROCK2 downstream signalling results in increase of phospho-S3 CFL1 in secretory endometrium (P < 0.001) as well as in vitro decidualized ESCs (P < 0.01) from controls but not PCOS patients. ROCK2-triggered alterations in the cytoskeleton are reflected by the significantly decreased motility of in vitro decidualized ESCs (P < 0.05). Proteomic and phosphoproteomic data are available via ProteomeXchange with identifier PXD026243. The number of biological samples was limited. The duration of protocol for isolation of non-cultured ESCs from tissue can potentially affect phosphorylation pathways in cells, yet the possible artefacts were minimized by the identical treatment of proliferative and secretory samples. The study demonstrated the benefits of combining the focussed kinase activity assay with wide-scale phosphoproteomics and showed the need for detailed elaboration of the in vitro decidualization protocols. ROCK was identified as the novel target of interest in decidualization, which requires closer attention in further studies-including the context of decidualization-related subfertility and infertility. This study was funded by the Estonian Ministry of Education and Research, and the Estonian Research Council (PRG1076, PRG454, PSG230 and PSG608), Enterprise Estonia (EU48695), Horizon 2020 innovation grant (ERIN, Grant no. EU952516) of the European Commission, the COMBIVET ERA Chair, H2020-WIDESPREAD-2018-04 (Grant agreement no. 857418), the Academy of Finland (Project grants 315921 and 321763), the Finnish Medical Foundation and The Sigrid Juselius Foundation. The authors confirm that they have no conflict of interest with respect to the content of this article.

Metal-Organic Framework-Derived Hollow and Hierarchical Porous Multivariate Metal-Oxide Microspheres for Efficient Phosphoproteomics Analysis.

Pre-enrichment of the biological samples is a crucial step in phosphoproteomics research. At present, metal-oxide affinity chromatography (MOAC) is one of the most recognized enrichment strategy. Therefore, the design and preparation of a MOAC-based affinity material with better enrichment properties will be of great significance for the phosphoproteomics study. In this work, we obtained a novel multivariate metal-oxide microsphere (NiFe2O4@C@TiO2) with a hollow and hierarchical porous structure through pyrolysis of TiO2-modified Fe/Ni-based metal-organic frameworks (MOFs). After pyrolysis, the carbon matrix derived from the MOFs provided support and porous properties. Meanwhile, multivariate metal oxides endowed the microspheres with an excellent magnetic response property and superior enrichment performance for phosphorylated biomolecules. The unique hollow and hierarchical porous structure greatly enhanced the diffusion of phosphorylated biomolecules. Therefore, the microspheres exhibited excellent enrichment performance for phosphorylated biomolecules: a large adsorption capacity (124 μmol g-1), excellent selectivity (α-casein/BSA, 1:5000, m/m), perfect size-exclusion performance (α-casein digests/α-casein/BSA, 1:500:500), and extremely low detection limit (2 fmol). Furthermore, the microspheres showed excellent enrichment performance in a series of real biological samples, such as nonfat milk, serum, saliva, rat brain tissue, and plasma exosomes of patients with esophageal cancer, which further demonstrated its huge application potential in MS-based phosphoproteomics research.

Integrated in silico MS-based phosphoproteomics and network enrichment analysis of RASopathy proteins

BackgroundRASopathies are a group of syndromes showing clinical overlap caused by mutations in genes affecting the RAS-MAPK pathway. Consequent disruption on cellular signaling leads and is driven by phosphoproteome remodeling. However, we still lack a comprehensive picture of the different key players and altered downstream effectors.MethodsAn in silico interactome of RASopathy proteins was generated using pathway enrichment analysis/STRING tool, including identification of main hub proteins. We also integrated phosphoproteomic and immunoblotting studies using previous published information on RASopathy proteins and their neighbors in the context of RASopathy syndromes. Data from Phosphosite database (www.phosphosite.org) was collected in order to obtain the potential phosphosites subjected to regulation in the 27 causative RASopathy proteins. We compiled a dataset of dysregulated phosphosites in RASopathies, searched for commonalities between syndromes in harmonized data, and analyzed the role of phosphorylation in the syndromes by the identification of key players between the causative RASopathy proteins and the associated interactome.ResultsIn this study, we provide a curated data set of 27 causative RASopathy genes, identify up to 511 protein–protein associations using pathway enrichment analysis/STRING tool, and identify 12 nodes as main hub proteins. We found that a large group of proteins contain tyrosine residues and their biological processes include but are not limited to the nervous system. Harmonizing published RASopathy phosphoproteomic and immunoblotting studies we identified a total of 147 phosphosites with increased phosphorylation, whereas 47 have reduced phosphorylation. The PKB signaling pathway is the most represented among the dysregulated phosphoproteins within the RASopathy proteins and their neighbors, followed by phosphoproteins implicated in the regulation of cell proliferation and the MAPK pathway.ConclusionsThis work illustrates the complex network underlying the RASopathies and the potential of phosphoproteomics for dissecting the molecular mechanisms in these syndromes. A combined study of associated genes, their interactome and phosphorylation events in RASopathies, elucidates key players and mechanisms to direct future research, diagnosis and therapeutic windows.

Structural analysis of mammalian protein phosphorylation at a proteome level

Phosphorylation is an essential post-translational modification for almost all cellular processes. Several global phosphoproteomics analyses have revealed phosphorylation profiles under different conditions. Beyond identification of phospho-sites, protein structures add another layer of information about their functionality. In this study, we systematically characterize phospho-sites based on their 3D locations in the protein and establish a location map for phospho-sites. More than 250,000 phospho-sites have been analyzed, of which 8,686 sites match at least one structure and are stratified based on their respective 3D positions. Core phospho-sites possess two distinct groups based on their dynamicity. Dynamic core phosphorylations are significantly more functional compared with static ones. The dynamic core and the interface phospho-sites are the most functional among all 3D phosphorylation groups. Our analysis provides global characterization and stratification of phospho-sites from a structural perspective that can be utilized for predicting functional relevance and filtering out false positives in phosphoproteomic studies.

Facile synthesis of bifunctional polymer monolithic column for tunable and specific capture of glycoproteins and phosphoproteins

Efficiently tunable capture of the glycosylated/phosphorylated proteins is critical to meet the need of in-depth glycoproteome and phosphoproteome studies. Reported here is a new bifunctional polymer monolithic column by introducing benzeneboronic acid and phosphonic acid onto monolithic column (denoted as poly (EDMA-co-VPBA-co-VPA) monolith) for tunable and specific enrichment of glycoproteins and phosphoproteins via switching different mobile phases. Based on boronate affinity and immobilized metal affinity, the as-prepared poly (EDMA-co-VPBA-co-VPA) monolith exhibited superior performance in selective separation of small molecules and biomacromolecules containing cis-diol/phosphate groups or not. And the frontal chromatography analysis showed that the binding capacity of the poly (EDMA-co-VPBA-co-VPA) monolith towards horseradish peroxidase (HRP, glycoprotein) or β-casein (phosphoprotein) is four-fold higher than that of bovine serum albumin (BSA, non-glycosylated/phosphorylated protein). Furthermore, combined with mass spectrometry identification, the successful application in specific enrichment of glycopeptides/phosphopeptides from tryptic digests of HRP/β-casein and direct capture of low abundant endogenous phosphopeptides from human serum proved great practicability in complex samples. This study provides a novel insight for fabricating the monolithic columns with multifunctionalization to facilitate further post-translational modification (PTM)-proteomics development.

A Two-Dimensional Metallacycle Cross-Linked Switchable Polymer for Fast and Highly Efficient Phosphorylated Peptide Enrichment.

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

One-step preparation of carbonaceous spheres rich in phosphate groups via hydrothermal carbonization for effective phosphopeptides enrichment

A green strategy was developed to prepare carbonaceous spheres rich in phosphoric acid groups on the surface with D-Glucose 6-phosphate sodium salt (called G6PNa2) as a sole carbon source through one-step hydrothermal carbonization method. The method is simple and facile and meets the standards of green chemistry as water is the sole solvent employed. Following the hydrothermal carbonization synthesis, the carbonaceous spheres were further functionalized with Ti4+. The main factors including reaction temperature, reaction time, and concentration of G6PNa2 were systematically studied in order to obtain the desirable morphology and the optimum phosphopeptides enrichment, for the resulting Ti4+ functionalized carbonaceous spheres (CS-Ti4+). The performance evaluation of the CS-Ti4+ prepared under the optimum conditions demonstrated excellent selectivity (1:1000), low detection limit (1 fmol) and high recovery rate (85%) towards phosphopeptides. Furthermore, 24 low-abundance phosphopeptides were captured from human saliva using CS-Ti4+, indicating its great potential in mass spectrometry-based phosphoproteome studies.

Global phosphoproteomics reveals DYRK1A regulates CDK1 activity in glioblastoma cells

Both tumour suppressive and oncogenic functions have been reported for dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Herein, we performed a detailed investigation to delineate the role of DYRK1A in glioblastoma. Our phosphoproteomic and mechanistic studies show that DYRK1A induces degradation of cyclin B by phosphorylating CDC23, which is necessary for the function of the anaphase-promoting complex, a ubiquitin ligase that degrades mitotic proteins. DYRK1A inhibition leads to the accumulation of cyclin B and activation of CDK1. Importantly, we established that the phenotypic response of glioblastoma cells to DYRK1A inhibition depends on both retinoblastoma (RB) expression and the degree of residual DYRK1A activity. Moderate DYRK1A inhibition leads to moderate cyclin B accumulation, CDK1 activation and increased proliferation in RB-deficient cells. In RB-proficient cells, cyclin B/CDK1 activation in response to DYRK1A inhibition is neutralized by the RB pathway, resulting in an unchanged proliferation rate. In contrast, complete DYRK1A inhibition with high doses of inhibitors results in massive cyclin B accumulation, saturation of CDK1 activity and cell cycle arrest, regardless of RB status. These findings provide new insights into the complexity of context-dependent DYRK1A signalling in cancer cells.

Phosphoproteome Study of Escherichia coli Devoid of Ser/Thr Kinase YeaG During the Metabolic Shift From Glucose to Malate.

Understanding phosphorylation-mediated regulation of metabolic enzymes, pathways, and cell phenotypes under metabolic shifts represents a major challenge. The kinases associated with most phosphorylation sites and the link between phosphorylation and enzyme activity remain unknown. In this study, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteome and phosphoproteome analysis of Escherichia coli ΔyeaG, a strain lacking a poorly characterized serine/threonine kinase YeaG, to decipher kinase-substrate interactions and the effects on metabolic phenotype during shifts from glucose to malate. The starting point of our analysis was the identification of physiological conditions under which ΔyeaG exhibits a clear phenotype. By metabolic profiling, we discovered that ΔyeaG strain has a significantly shorter lag phase than the wild type during metabolic shift from glucose to malate. Under those conditions, our SILAC analysis revealed several proteins that were differentially phosphorylated in the ΔyeaG strain. By focusing on metabolic enzymes potentially involved in central carbon metabolism, we narrowed down our search for putative YeaG substrates and identified isocitrate lyase AceA as the direct substrate of YeaG. YeaG was capable of phosphorylating AceA in vitro only in the presence of malate, suggesting that this phosphorylation event is indeed relevant for glucose to malate shift. There is currently not enough evidence to firmly establish the exact mechanism of this newly observed regulatory phenomenon. However, our study clearly exemplifies the usefulness of SILAC-based approaches in identifying proteins kinase substrates, when applied in physiological conditions relevant for the activity of the protein kinase in question.