Proteome and Phosphoproteome Profiling Reveal the Toxic Mechanism of Clostridium perfringens Epsilon Toxin in MDCK Cells.

Epsilon toxin (ETX), a potential agent of biological and toxic warfare, causes the death of many ruminants and threatens human health. It is crucial to understand the toxic mechanism of such a highly lethal and rapid course toxin. In this study, we detected the effects of ETX on the proteome and phosphoproteome of MDCK cells after 10 min and 30 min. A total of 44 differentially expressed proteins (DEPs) and 588 differentially phosphorylated proteins (DPPs) were screened in the 10 min group, while 73 DEPs and 489 DPPs were screened in the 30 min group. ETX-induced proteins and phosphorylated proteins were mainly located in the nucleus, cytoplasm, and mitochondria, and their enrichment pathways were related to transcription and translation, virus infection, and intercellular junction. Meanwhile, the protein-protein interaction network screened out several hub proteins, including SRSF1/2/6/7/11, SF3B1/2, NOP14/56, ANLN, GTPBP4, THOC2, and RRP1B. Almost all of these proteins were present in the spliceosome pathway, indicating that the spliceosome pathway is involved in ETX-induced cell death. Next, we used RNAi lentiviruses and inhibitors of several key proteins to verify whether these proteins play a critical role. The results confirmed that SRSF1, SF3B2, and THOC2 were the key proteins involved in the cytotoxic effect of ETX. In addition, we found that the common upstream kinase of these key proteins was SRPK1, and a reduction in the level of SRPK1 could also reduce ETX-induced cell death. This result was consistent with the phosphorylated proteomics analysis. In summary, our study demonstrated that ETX induces phosphorylation of SRSF1, SF3B2, THOC2, and SRPK1 proteins on the spliceosome pathway, which inhibits normal splicing of mRNA and leads to cell death.

Broad proteomics analysis of seeding-induced aggregation of α-synuclein in M83 neurons reveals remodeling of proteostasis mechanisms that might contribute to Parkinson's disease pathogenesis.

Aggregation of misfolded α-synuclein (α-syn) is a key characteristic feature of Parkinson's disease (PD) and related synucleinopathies. The nature of these aggregates and their contribution to cellular dysfunction is still not clearly elucidated. We employed mass spectrometry-based total and phospho-proteomics to characterize the underlying molecular and biological changes due to α-syn aggregation using the M83 mouse primary neuronal model of PD. We identified gross changes in the proteome that coincided with the formation of large Lewy body-like α-syn aggregates in these neurons. We used protein-protein interaction (PPI)-based network analysis to identify key protein clusters modulating specific biological pathways that may be dysregulated and identified several mechanisms that regulate protein homeostasis (proteostasis). The observed changes in the proteome may include both homeostatic compensation and dysregulation due to α-syn aggregation and a greater understanding of both processes and their role in α-syn-related proteostasis may lead to improved therapeutic options for patients with PD and related disorders.

Illuminating phenotypic drug responses of sarcoma cells to kinase inhibitors by phosphoproteomics.

Kinase inhibitors (KIs) are important cancer drugs but often feature polypharmacology that is molecularly not understood. This disconnect is particularly apparent in cancer entities such as sarcomas for which the oncogenic drivers are often not clear. To investigate more systematically how the cellular proteotypes of sarcoma cells shape their response to molecularly targeted drugs, we profiled the proteomes and phosphoproteomes of 17 sarcoma cell lines and screened the same against 150 cancer drugs. The resulting 2550 phenotypic profiles revealed distinct drug responses and the cellular activity landscapes derived from deep (phospho)proteomes (9-10,000 proteins and 10-27,000 phosphorylation sites per cell line) enabled several lines of analysis. For instance, connecting the (phospho)proteomic data with drug responses revealed known and novel mechanisms of action (MoAs) of KIs and identified markers of drug sensitivity or resistance. All data is publicly accessible via an interactive web application that enables exploration of this rich molecular resource for a better understanding of active signalling pathways in sarcoma cells, identifying treatment response predictors and revealing novel MoA of clinical KIs.

Phosphoproteome analyses pinpoint the F-box protein SLOW MOTION as a regulator of warm temperature-mediated hypocotyl growth in Arabidopsis.

Hypocotyl elongation is controlled by several signals and is a major characteristic of plants growing in darkness or under warm temperature. While already several molecular mechanisms associated with this process are known, protein degradation and associated E3 ligases have hardly been studied in the context of warm temperature. In a time-course phosphoproteome analysis on Arabidopsis seedlings exposed to control or warm ambient temperature, we observed reduced levels of diverse proteins over time, which could be due to transcription, translation, and/or degradation. In addition, we observed differential phosphorylation of the LRR F-box protein SLOMO MOTION (SLOMO) at two serine residues. We demonstrate that SLOMO is a negative regulator of hypocotyl growth, also under warm temperature conditions, and protein-protein interaction studies revealed possible interactors of SLOMO, such as MKK5, DWF1, and NCED4. We identified DWF1 as a likely SLOMO substrate and a regulator of warm temperature-mediated hypocotyl growth. We propose that warm temperature-mediated regulation of SLOMO activity controls the abundance of hypocotyl growth regulators, such as DWF1, through ubiquitin-mediated degradation.

Proteostatic modulation in brain aging without associated Alzheimer's disease-and age-related neuropathological changes.

AIMS: (Phospho)proteomics of old-aged subjects without cognitive or behavioral symptoms, and without AD-neuropathological changes and lacking any other neurodegenerative alteration will increase understanding about the physiological state of human brain aging without associate neurological deficits and neuropathological lesions. METHODS: (Phospho)proteomics using conventional label-free- and SWATH-MS (Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) has been assessed in the frontal cortex (FC) of individuals without NFTs, senile plaques (SPs) and age-related co-morbidities classified by age (years) in four groups; group 1 (young, 30-44); group 2 (middle-aged: MA, 45-52); group 3 (early-elderly, 64-70); and group 4 (late-elderly, 75-85). RESULTS: Protein levels and deregulated protein phosphorylation linked to similar biological terms/functions, but involving different individual proteins, are found in FC with age. The modified expression occurs in cytoskeleton proteins, membranes, synapses, vesicles, myelin, membrane transport and ion channels, DNA and RNA metabolism, ubiquitin-proteasome-system (UPS), kinases and phosphatases, fatty acid metabolism, and mitochondria. Dysregulated phosphoproteins are associated with the cytoskeleton, including microfilaments, actin-binding proteins, intermediate filaments of neurons and glial cells, and microtubules; membrane proteins, synapses, and dense core vesicles; kinases and phosphatases; proteins linked to DNA and RNA; members of the UPS; GTPase regulation; inflammation; and lipid metabolism. Noteworthy, protein levels of large clusters of hierarchically-related protein expression levels are stable until 70. However, protein levels of components of cell membranes, vesicles and synapses, RNA modulation, and cellular structures (including tau and tubulin filaments) are markedly altered from the age of 75. Similarly, marked modifications occur in the larger phosphoprotein clusters involving cytoskeleton and neuronal structures, membrane stabilization, and kinase regulation in the late elderly. CONCLUSIONS: Present findings may increase understanding of human brain proteostasis modifications in the elderly in the subpopulation of individuals not having AD neuropathological change and any other neurodegenerative change in any telencephalon region.

Reverse Phase Protein Arrays in cancer stem cells.

The scenario of proteogenomics is rapidly evolving and novel technologies are enabling comprehensive molecular exploration down to single cells. Likewise, digital (immuno-)assays are revolutionizing the field of biomarker detection and have reached the grade for population-level screenings with single-molecule sensitivity. Nonetheless, cost- and time-effective, high-throughput targeted phospho-proteomics at a preclinical stage still relies on ad hoc microarray platforms, such as the Reverse-Phase Protein microArrays (RPPA). Although this technique requires specific knowledge and equipment and different laboratories worldwide have implemented alternative methodological strategies, the application of RPPA to biomarker discovery has proven successful on diverse types of samples, including tissues and biological fluids as well as nanovesicles and in vitro cultured lines. Among these, cancer stem(-like) cells (CSC) represent an ideal experimental model system for preclinical discovery and definition of novel drug targets. The present methodological article provides the basic knowledge and steps on how to deploy an RPPA analysis with specific reference to an ideal experimental setup of drug testing on CSC.

Dysregulated Protein Phosphorylation in a Mouse Model of FTLD-Tau.

The neocortex of P301S mice, used as a model of fronto-temporal lobar degeneration linked to tau mutation (FTLD-tau), and wild-type mice, both aged 9 months, were analyzed with conventional label-free phosphoproteomics and SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) to assess the (phospho)proteomes. The total number of identified dysregulated phosphoproteins was 328 corresponding to 524 phosphorylation sites. The majority of dysregulated phosphoproteins, most of them hyperphosphorylated, were proteins of the membranes, synapses, membrane trafficking, membrane vesicles linked to endo- and exocytosis, cytoplasmic vesicles, and cytoskeleton. Another group was composed of kinases. In contrast, proteins linked to DNA, RNA metabolism, RNA splicing, and protein synthesis were hypophosphorylated. Other pathways modulating energy metabolism, cell signaling, Golgi apparatus, carbohydrates, and lipids are also targets of dysregulated protein phosphorylation in P301S mice. The present results, together with accompanying immunohistochemical and Western-blotting studies, show widespread abnormal phosphorylation of proteins, in addition to protein tau, in P301S mice. These observations point to dysregulated protein phosphorylation as a relevant contributory pathogenic component of tauopathies.

Phospho-proteomics reveals that RSK signaling is required for proliferation of natural killer cells stimulated with IL-2 or IL-15.

Natural killer (NK) cells are cytotoxic lymphocytes that play a critical role in the innate immune system. Although cytokine signaling is crucial for the development, expansion, and cytotoxicity of NK cells, the signaling pathways stimulated by cytokines are not well understood. Here, we sought to compare the early signaling dynamics induced by the cytokines interleukin (IL)-2 and IL-15 using liquid chromatography-mass spectrometry (LC-MS)-based phospho-proteomics. Following stimulation of the immortalized NK cell line NK-92 with IL-2 or IL-15 for 5, 10, 15, or 30 min, we identified 8,692 phospho-peptides from 3,023 proteins. Comparing the kinetic profiles of 3,619 fully quantified phospho-peptides, we found that IL-2 and IL-15 induced highly similar signaling in NK-92 cells. Among the IL-2/IL-15-regulated phospho-peptides were both well-known signaling events like the JAK/STAT pathway and novel signaling events with potential functional significance including LCP1 pSer5, STMN1 pSer25, CHEK1 pSer286, STIM1 pSer608, and VDAC1 pSer104. Using bioinformatic approaches, we sought to identify kinases regulated by IL-2/IL-15 stimulation and found that the p90 ribosomal S6 kinase (p90RSK) family was activated by both cytokines. Using pharmacological inhibitors, we then discovered that RSK signaling is required for IL-2 and IL-15-induced proliferation in NK-92 cells. Taken together, our analysis represents the first phospho-proteomic characterization of cytokine signaling in NK cells and increases our understanding of how cytokine signaling regulates NK cell function.

Novel Antibody-Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma.

We investigated the feasibility of detecting the presence of specific autoantibodies against potential tumor-associated peptide antigens by enriching these antibody-peptide complexes using Melon Gel resin and mass spectrometry. Our goal was to find tumor-associated phospho-sites that trigger immunoreactions and raise autoantibodies that are detectable in plasma of glioma patients. Such immunoglobulins can potentially be used as targets in immunotherapy. To that aim, we describe a method to detect the presence of antibodies in biological samples that are specific to selected clinically relevant peptides. The method is based on the formation of antibody-peptide complexes by mixing patient plasma with a glioblastoma multiforme (GBM) derived peptide library, enrichment of antibodies and antibody-peptide complexes, the separation of peptides after they are released from immunoglobulins by molecular weight filtration and finally mass spectrometric quantification of these peptides. As proof of concept, we successfully applied the method to dinitrophenyl (DNP)-labeled α-casein peptides mixed with anti-DNP. Further, we incubated human plasma with a phospho-peptide library and conducted targeted analysis on EGFR and GFAP phospho-peptides. As a result, immunoaffinity against phospho-peptide GSHQIS[+80]LDNPDYQQDFFPK (EGFR phospho-site S1166) was detected in high-grade glioma (HGG) patient plasma but not in healthy donor plasma. For the GFAP phospho-sites selected, such immunoaffinity was not observed.

Differential Phospho-Signatures in Blood Cells Identify LRRK2 G2019S Carriers in Parkinson's Disease.

BACKGROUND: The clinicopathological phenotype of G2019S LRRK2-associated Parkinson's disease (L2PD) is similar to idiopathic Parkinson's disease (iPD), and G2019S LRRK2 nonmanifesting carriers (L2NMCs) are at increased risk for development of PD. With various therapeutic strategies in the clinical and preclinical pipeline, there is an urgent need to identify biomarkers that can aid early diagnosis and patient enrichment for ongoing and future LRRK2-targeted trials. OBJECTIVE: The objective of this work was to investigate differential protein and phospho-protein changes related to G2019S mutant LRRK2 in peripheral blood mononuclear cells from G2019S L2PD patients and G2019S L2NMCs, identify specific phospho-protein changes associated with the G2019S mutation and with disease status, and compare findings with patients with iPD. METHODS: We performed an unbiased phospho-proteomic study by isobaric label-based mass spectrometry using peripheral blood mononuclear cell group pools from a LRRK2 cohort from Spain encompassing patients with G2019S L2PD (n = 20), G2019S L2NMCs (n = 20), healthy control subjects (n = 30), patients with iPD (n = 15), patients with R1441G L2PD (n = 5), and R1441G L2NMCs (n = 3) (total N = 93). RESULTS: Comparing G2019S carriers with healthy controls, we identified phospho-protein changes associated with the G2019S mutation. Moreover, we uncovered a specific G2019S phospho-signature that changes with disease status and can discriminate patients with G2019S L2PD, G2019S L2NMCs, and healthy controls. Although patients with iPD showed a differential phospho-proteomic profile, biological enrichment analyses revealed similar changes in deregulated pathways across the three groups. CONCLUSIONS: We found a differential phospho-signature associated with LRRK2 G2019S for which, consistent with disease status, the phospho-profile from PD at-risk G2019S L2NMCs was more similar to healthy controls than patients with G2019S L2PD with the manifested disease. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The Arabidopsis Root Tip (Phospho)Proteomes at Growth-Promoting versus Growth-Repressing Conditions Reveal Novel Root Growth Regulators.

Auxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies have revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different concentrations of the synthetic auxin NAA that establish growth-promoting and -repressing conditions on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data, we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results, together with previously published studies, suggest that auxin, H+-ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr31 phosphorylation site for growth regulation in the Arabidopsis root tip.

A phospho-proteomic study of cetuximab resistance in KRAS/NRAS/BRAFV600 wild-type colorectal cancer.

PURPOSE: We hypothesised that plasticity in signal transduction may be a mechanism of drug resistance and tested this hypothesis in the setting of cetuximab resistance in patients with KRAS/NRAS/BRAFV600 wild-type colorectal cancer (CRC). METHODS: A multiplex antibody-based platform was used to study simultaneous changes in signal transduction of 55 phospho-proteins in 12 KRAS/NRAS/BRAFV600 wild-type CRC cell lines (6 cetuximab sensitive versus 6 cetuximab resistant) following 1 and 4 h in vitro cetuximab exposure. We validated our results in CRC patient samples (n = 4) using ex vivo exposure to cetuximab in KRAS/NRAS/BRAFV600 cells that were immunomagnetically separated from the serous effusions of patients with known cetuximab resistance. RESULTS: Differences in levels of phospho-proteins in cetuximab sensitive and resistant cell lines included reductions in phospho-RPS6 and phospho-PRAS40 in cetuximab sensitive, but not cetuximab resistant cell lines at 1 and 4 h, respectively. In addition, phospho-AKT levels were found to be elevated in 3/4 patient samples following ex vivo incubation with cetuximab for 1 h. We further explored these findings by studying the effects of combinations of cetuximab and two PI3K pathway inhibitors in 3 cetuximab resistant cell lines. The addition of PI3K pathway inhibitors to cetuximab led to a significantly higher reduction in colony formation capacity compared to cetuximab alone. CONCLUSION: Our findings suggest activation of the PI3K pathway as a mechanism of cetuximab resistance in KRAS/NRAS/BRAFV600 wild-type CRC.

Triple extraction method enables high quality mass spectrometry-based proteomics and phospho-proteomics for eventual multi-omics integration studies.

Large-scale multi-omic analysis allows a thorough understanding of different physiological or pathological conditions, particularly cancer. Here, an extraction method simultaneously yielding DNA, RNA and protein (thereby referred to as "triple extraction", TEx) was tested for its suitability to unbiased, system-wide proteomic investigation. Largely proven efficient for transcriptomic and genomic studies, we aimed at exploring TEx compatibility with mass spectrometry-based proteomics and phospho-proteomics, as compared to a standard urea extraction. TEx is suitable for the shotgun investigation of proteomes, providing similar results as urea-based protocol both at the qualitative and quantitative levels. TEx is likewise compatible with the exploration of phosphorylation events, actually providing a higher number of correctly localized sites than urea, although the nature of extracted modifications appears somewhat distinct between both techniques. These results highlight that the presented protocol is well suited for the examination of the proteome and modified proteome of this bladder cancer cell model, as efficiently as other more widely used workflows for mass spectrometry-based analysis. Potentially applicable to other mammalian cell types and tissues, TEx represents an advantageous strategy for multi-omics on scarce and/or heterogenous samples.

Dysregulated protein phosphorylation: A determining condition in the continuum of brain aging and Alzheimer's disease.

Tau hyperphosphorylation is the first step of neurofibrillary tangle (NFT) formation. In the present study, samples of the entorhinal cortex (EC) and frontal cortex area 8 (FC) of cases with NFT pathology classified as stages I-II, III-IV, and V-VI without comorbidities, and of middle-aged (MA) individuals with no NFT pathology, were analyzed by conventional label-free and SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) to assess the (phospho)proteomes. The total number of identified dysregulated phosphoproteins was 214 in the EC, 65 of which were dysregulated at the first stages (I-II) of NFT pathology; 167 phosphoproteins were dysregulated in the FC, 81 of them at stages I-II of NFT pathology. A large percentage of dysregulated phosphoproteins were identified in the two regions and at different stages of NFT progression. The main group of dysregulated phosphoproteins was made up of components of the membranes, cytoskeleton, synapses, proteins linked to membrane transport and ion channels, and kinases. The present results show abnormal phosphorylation of proteins at the first stages of NFT pathology in the elderly (in individuals clinically considered representative of normal aging) and sporadic Alzheimer's disease (sAD). Dysregulated protein phosphorylation in the FC precedes the formation of NFTs and SPs. The most active period of dysregulated phosphorylation is at stages III-IV when a subpopulation of individuals might be clinically categorized as suffering from mild cognitive impairment which is a preceding determinant stage in the progression to dementia. Altered phosphorylation of selected proteins, carried out by activation of several kinases, may alter membrane and cytoskeletal functions, among them synaptic transmission and membrane/cytoskeleton signaling. Besides their implications in sAD, the present observations suggest a molecular substrate for "benign" cognitive deterioration in "normal" brain aging.

Integrated Proteomic and Phosphoproteomics Analysis of DKK3 Signaling Reveals Activated Kinase in the Most Aggressive Gallbladder Cancer.

DKK3 is a secreted protein, which belongs to a family of Wnt antagonists and acts as a potential tumor suppressor in gallbladder cancer. To further understand its tumor suppressor functions, we overexpressed DKK3 in 3 GBC cell lines. We have employed high-resolution mass spectrometry and tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography to enrich phosphopeptides to check the downstream regulators. In this study, we reported for the first time the alteration in the phosphorylation of 14 kinases upon DKK3 overexpression. In addition, we observed DKK3 induced hyper phosphorylation of 2 phosphatases: PPP1R12A and PTPRA, which have not been reported previously. Canonical pathway analysis of altered molecules indicated differential enrichment of signaling cascades upon DKK3 overexpression in all the 3 cell lines. Protein kinase A signaling, Sirtuin signaling pathway, and Cell Cycle Control of Chromosomal Replication were observed to be differentially activated in the GBC cell lines. Our study revealed, DKK3 overexpression has differential effect based on the aggressive behavior of the cell lines. This study expands the understanding of DKK3-mediated signaling events and can be used as a primary factor for understanding the complex nature of this molecule.

Feasibility of phosphoproteomics to uncover oncogenic signalling in secreted extracellular vesicles using glioblastoma-EGFRVIII cells as a model.

Cancer cells secrete extracellular vesicles (EVs) that contain molecular information, including proteins and RNA. Oncogenic signalling can be transferred via the cargo of EVs to recipient cells and may influence the behaviour of neighbouring cells or cells at a distance. This cargo may contain cancer drivers, such as EGFR, and also phosphorylated (activated) components of oncogenic signalling cascades. Till date, the cancer EV phosphoproteome has not been studied in great detail. In the present study, we used U87 and U87EGFRvIII cells as a model to explore EV oncogenic signalling components in comparison to the cellular profile. EVs were isolated using the VN96 ME-kit and subjected to LC-MS/MS based phosphoproteomics and dedicated bioinformatics. Expression of (phosphorylated)-EGFR was highly increased in EGFRvIII overexpressing cells and their secreted EVs. The increased phosphorylated proteins in both cells and EVs were associated with activated components of the EGFR-signalling cascade and included EGFR, AKT2, MAPK8, SMG1, MAP3K7, DYRK1A, RPS6KA3 and PAK4 kinases. In conclusion, EVs harbour oncogenic signalling networks including multiple activated kinases including EGFR, AKT and mTOR. SIGNIFICANCE: Extracellular vesicles (EVs) are biomarker treasure troves and are widely studied for their biomarker content in cancer. However, little research has been done on the phosphorylated protein profile within cancer EVs. In the current study, we demonstrate that EVs that are secreted by U87-EGFRvIII mutant glioblastoma cells contain high levels of oncogenic signalling networks. These networks contain multiple activated (phosphorylated) kinases, including EGFR, MAPK, AKT and mTOR.

Integrative proteomics reveals principles of dynamic phosphosignaling networks in human erythropoiesis.

Human erythropoiesis is an exquisitely controlled multistep developmental process, and its dysregulation leads to numerous human diseases. Transcriptome and epigenome studies provided insights into system-wide regulation, but we currently lack a global mechanistic view on the dynamics of proteome and post-translational regulation coordinating erythroid maturation. We established a mass spectrometry (MS)-based proteomics workflow to quantify and dynamically track 7,400 proteins and 27,000 phosphorylation sites of five distinct maturation stages of in vitro reconstituted erythropoiesis of CD34+ HSPCs. Our data reveal developmental regulation through drastic proteome remodeling across stages of erythroid maturation encompassing most protein classes. This includes various orchestrated changes in solute carriers indicating adjustments to altered metabolic requirements. To define the distinct proteome of each maturation stage, we developed a computational deconvolution approach which revealed stage-specific marker proteins. The dynamic phosphoproteomes combined with a kinome-targeted CRISPR/Cas9 screen uncovered coordinated networks of erythropoietic kinases and pinpointed downregulation of c-Kit/MAPK signaling axis as key driver of maturation. Our system-wide view establishes the functional dynamic of complex phosphosignaling networks and regulation through proteome remodeling in erythropoiesis.

Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Quantitative Mass-Spectrometry.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) within proteins have attracted considerable attention in recent years. Several important biological signaling mechanisms including protein-protein interactions and post-translational modifications can be easily mediated by IDPs and IDRs due to their flexible structure. These regions can encode linear sequences that are indispensable in cell-signaling networks and circuits. For example, the linear multisite phosphorylation networks encoded in disordered protein sequences play a key role in cell-cycle regulation where the phosphorylation of proteins controls the orchestration of all major mechanisms. While elucidating a systems-level understanding of this process and other multisite phosphorylation processes, we extensively used mass-spectrometry and found it to be an ideal tool to identify, characterize, and quantify phosphorylation dynamics within IDPs. Here, we describe a quantitative proteomics method, together with a detailed protocol to analyze dynamic multisite phosphorylation processes within IDPs using an in vitro protein phosphorylation assay with "light" gamma-16O ATP and "heavy" gamma-18O ATP, combined with liquid chromatography mass spectrometry.

Phospho-PTM proteomic discovery of novel EPO- modulated kinases and phosphatases, including PTPN18 as a positive regulator of EPOR/JAK2 Signaling.

The formation of erythroid progenitor cells depends sharply upon erythropoietin (EPO), its cell surface receptor (erythropoietin receptor, EPOR), and Janus kinase 2 (JAK2). Clinically, recombinant human EPO (rhEPO) additionally is an important anti-anemia agent for chronic kidney disease (CKD), myelodysplastic syndrome (MDS) and chemotherapy, but induces hypertension, and can exert certain pro-tumorigenic effects. Cellular signals transduced by EPOR/JAK2 complexes, and the nature of EPO-modulated signal transduction factors, therefore are of significant interest. By employing phospho-tyrosine post-translational modification (p-Y PTM) proteomics and human EPO- dependent UT7epo cells, we have identified 22 novel kinases and phosphatases as novel EPO targets, together with their specific sites of p-Y modification. New kinases modified due to EPO include membrane palmitoylated protein 1 (MPP1) and guanylate kinase 1 (GUK1) guanylate kinases, together with the cytoskeleton remodeling kinases, pseudopodium enriched atypical kinase 1 (PEAK1) and AP2 associated kinase 1 (AAK1). Novel EPO- modified phosphatases include protein tyrosine phosphatase receptor type A (PTPRA), phosphohistidine phosphatase 1 (PHPT1), tensin 2 (TENC1), ubiquitin associated and SH3 domain containing B (UBASH3B) and protein tyrosine phosphatase non-receptor type 18 (PTPN18). Based on PTPN18's high expression in hematopoietic progenitors, its novel connection to JAK kinase signaling, and a unique EPO- regulated PTPN18-pY389 motif which is modulated by JAK2 inhibitors, PTPN18's actions in UT7epo cells were investigated. Upon ectopic expression, wt-PTPN18 promoted EPO dose-dependent cell proliferation, and survival. Mechanistically, PTPN18 sustained the EPO- induced activation of not only mitogen-activated protein kinases 1 and 3 (ERK1/2), AKT serine/threonine kinase 1-3 (AKT), and signal transducer and activator of transcription 5A and 5B (STAT5), but also JAK2. Each effect further proved to depend upon PTPN18's EPO- modulated (p)Y389 site. In analyses of the EPOR and the associated adaptor protein RHEX (regulator of hemoglobinization and erythroid cell expansion), wt-PTPN18 increased high molecular weight EPOR forms, while sharply inhibiting the EPO-induced phosphorylation of RHEX-pY141. Each effect likewise depended upon PTPN18-Y389. PTPN18 thus promotes signals for EPO-dependent hematopoietic cell growth, and may represent a new druggable target for myeloproliferative neoplasms.

Targeting proline in (phospho)proteomics.

Mass spectrometry-based proteomics experiments typically start with the digestion of proteins using trypsin, chosen because of its high specificity, availability, and ease of use. It has become apparent that the sole use of trypsin may impose certain limits on our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments, or even subsets of proteins. To tackle this problem, alternative proteases have been introduced and shown to lead to an increase in the detectable (phospho)proteome. Here, we argue that there may be further room for improvement and explore the protease EndoPro. For optimal peptide identification rates, we explored multiple peptide fragmentation techniques (HCD, ETD, and EThcD) and employed Byonic as search algorithm. We obtain peptide IDs for about 40% of the MS2 spectra (66% for trypsin). EndoPro cleaves with high specificity at the C-terminal site of Pro and Ala residues and displays activity in a broad pH range, where we focused on its performance at pH = 2 and 5.5. The proteome coverage of EndoPro at these two pH values is rather distinct, and also complementary to the coverage obtained with trypsin. As about 40% of mammalian protein phosphorylations are proline-directed, we also explored the performance of EndoPro in phosphoproteomics. EndoPro extends the coverable phosphoproteome substantially, whereby both the, at pH = 2 and 5.5, acquired phosphoproteomes are complementary to each other and to the phosphoproteome obtained using trypsin. Hence, EndoPro is a powerful tool to exploit in (phospho)proteomics applications.