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Phosphoproteomics sample preparation impacts biological interpretation of signalling responses

ABSTRACT: Quantitative data-dependent acquisition phosphoproteomics (DDAP) has revolutionised cell signaling studies. However, the impact of the specific DDAP sample preparation method on the phosphoproteome quantified is unclear as each study quantifies only a fraction of the phosphoproteome. Here, we compared two distinct phosphoproteome datasets profiled either by a simple or a complex workflow. The complex workflow involved a strong-cation exchange chromatography-based phosphoproteomics (SCXPhos) and the simple workflow involved a modified single-run high-throughput EasyPhos-based phosphoproteomics (HighPhos). We used mouse embryonic stem cells as a biological system for the comparison and profiled phosphoproteomes at two different time points after exposure to two different doses of ionizing radiation (IR). Both methods achieved equal coverage of ~20,000 phosphosites in total, whereas combined profiling resulted in a dramatic increase in the depth of the phosphoproteome (>30,000 phosphosites). Over 96% of the identified IR-responsive phosphosites were method-specific. Despite such limited overlap, both methods uncovered a prominent role for ATM-mediated phosphorylation and reproducibly quantified a subset of shared IR-responsive phosphosites. The majority of IR-responsive phosphosites identified by SCXPhos concerned single phosphorylation events, whereas double- and multi-site phosphorylation events predominated after HighPhos. The coverage of the phosphoproteome increased to >82,000 phosphosites when multiple datasets were included suggesting that current global phosphoproteome studies are nowhere near representing the entire spectrum of phosphorylation events. Thus, our results demonstrate the power of the combination of different methodologies to obtain a better representative image of a broad view of phosphorylation signaling events.

INSTRUMENT(S): Q Exactive HF, Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Embryonic Stem Cell

SUBMITTER: Bharath Sampadi

LAB HEAD: Harry Vrieling

PROVIDER: PXD025376 | Pride | 2021-12-25

REPOSITORIES: Pride

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Publications

Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes.

Sampadi Bharath B Mullenders Leon H F LHF Vrieling Harry H

Cells 20211203 12

The influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling targets uncovered by comparing the phosphoproteomes generated by two commonly used methods-strong cation exchange chromatography-based phosphoproteomics (SCXPhos) and single-run high-throughput phosphoproteomics (HighPhos). Phosphoproteomes of embryonic stem cells exposed to ionizing radiation (IR) profiled ...[more]

PMID: 34943915

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Project description:The rapid activation of RAF-SnRK2 cascades in response to hyperosmolarity is a crucial event in plants' adaptation to osmotic stress, yet the underlying mechanisms have remained elusive. In this study, we reveal that the calcium chelator EGTA, but not other calcium modulators, can potently activate both RAFs and SnRK2s. We demonstrate that EGTA induces the phosphorylation of RAF24, serving as an indicator of rapid RAF activation. Furthermore, we show that EGTA treatment results in increased phosphorylation of Ser171 and Ser175 in SnRK2.6, known markers of SnRK2 activation. In-gel kinase assays confirmed the activation of RAFs and SnRK2s. These findings collectively highlight that exogenous EGTA application mimics mannitol treatment, effectively triggering the activation of both RAF and SnRK2 subfamily kinases. Utilizing a DIA-based phosphoproteomics approach, we profiled global phosphorylation changes in response to EGTA and mannitol treatments. We identified 22,991 phosphorylated peptides corresponding to 6,515 phosphosites on 1,790 proteins. Notably, 5,067 sites were classified as class I phosphorylation sites, indicating high confidence in their localization. Principal Component Analysis confirmed the distinct phosphoproteomic profiles of the three conditions, with excellent reproducibility among biological replicates. EGTA treatment induced substantial changes in global phosphorylation, with 3,987 phosphosites increased and 964 decreased, signifying a profound impact on protein phosphorylation. The activation of RAFs and SnRK2s was supported by significant phosphorylation changes in these kinases. Additionally, EGTA-induced phosphoproteins included downstream effectors of RAF-SnRK2 cascades. GO analysis revealed distinct functional categories for EGTA and mannitol-responsive phosphoproteins, shedding light on the divergent roles of these treatments. Our study provides novel insights into the role of EGTA in the activation of RAF-SnRK2 cascades and its broader impact on the plant phosphoproteome, offering a valuable resource for understanding the downstream events related to exocellular calcium depletion in plants. It also highlights the potential involvement of MAP4Ks and resistosome-related kinases in osmotic stress signaling. Overall, our research enhances our understanding of osmotic stress response mechanisms and the power of DIA-based phosphoproteomics in unraveling complex phosphorylation events in plants.

Project description:Background Annexin-1 (ANXA1) plays pivotal roles in regulating various physiological processes including inflammation, proliferation and apoptosis, and deregulations of ANXA1 functions have been associated with tumorigenesis and metastasis events in several cancers. Though ANXA1 levels correlate with breast cancer disease status and outcome, its distinct functional involvement in breast cancer initiation and progression remains unclear. We hypothesized ANXA1-responsive kinase signaling alteration and associated phosphorylation signaling underlie early events in breast cancer initiation events and hence profiled ANXA1-dependent phosphorylation changes in mammary gland epithelial cells. Methods Quantitative phosphoproteomics analysis of mammary gland epithelial cells derived from ANXA1-heterozygous and ANXA1-deficient mice was carried out using SILAC-based mass spectrometry. Kinase and signaling changes underlying ANXA1 perturbations were derived by upstream kinase prediction and integrated network analysis of altered proteins and phosphoproteins. Results We identified a total of 8,110 unique phosphorylation sites, of which 582 phosphorylation sites on 372 proteins showed ANXA1-responsive changes. A majority of these phosphorylation changes occurred on proteins associated with cytoskeletal reorganization spanning the focal adhesion, stress fibers, and also the microtubule network pressing on new roles for ANXA1 in regulating microtubule dynamics. Comparative analysis of regulated global proteome and phosphoproteome highlighted key differences in translational and post-translational effects of ANXA1, and stressed on a close-coordinated rewiring of the cell adhesion network. Kinase prediction analysis suggested activity modulation of CAMK2, PAK, ERK, and IKK kinases upon loss of ANXA1. Integrative analysis revealed regulation of WNT and also Hippo signaling pathways in ANXA1-deficient mammary epithelial cells, wherein there is a downregulation of transcriptional effects of TEAD suggestive of ANXA1-responsive transcriptional rewiring. Conclusions The phosphoproteome landscape uncovered several novel perspectives for ANXA1 in mammary gland biology and stressed on its involvement in key signaling pathways modulating cell adhesion and migration that could contribute to breast cancer initiation.

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Project description:Protein phosphorylation regulated by plant hormone is involved in the coordination of fundamental plant development. Brassinosteroids (BRs), a group of phytohormones, regulated phosphorylation dynamics remains to be delineated in plants. In this study, we performed a mass spectrometry (MS)-based phosphoproteomics to conduct a global and dynamic phosphoproteome profiling across five time points of BR treatment in the period between 5 minutes and 12 hours. MS coupling with phosphopeptide enrichment techniques has become the powerful tool for profiling protein phosphorylation. However, MS-based methods tend to have data consistency and coverage issues. To address these issues, bioinformatics approaches were used to complement the non-detected proteins and recover the dynamics of phosphorylation events. A total of 1,104 unique phosphorylated peptides from 739 unique phosphoproteins were identified. The time-dependent gene ontology (GO) analysis shows the transition of biological processes from signaling transduction to morphogenesis and stress response. The protein-protein interaction analysis found most of identified phosphoproteins have strongly connections with known BR signaling components. The analysis by using Motif-X was performed to identify 15 enriched motifs, 11 of which correspond to 6 known kinase families. To uncover the dynamic activities of kinases, the enriched motifs were combined with phosphorylation profiles and revealed that the substrates of casein kinase 2 and mitogen-activated protein kinase were significantly phosphorylated and dephosphorylated at initial time of BR treatment, respectively. The time-dependent kinase-substrate interaction networks were constructed and showed many substrates are the downstream of other signalings, such as auxin and ABA signalings. Comparing BR responsive phosphoproteome and gene expression data, we found most of phosphorylation changes were not led by gene expression changes. Our results suggested BR signaling not only induces gene expression, but also change protein activation by phosphorylation via various kinases. Through a large-scale dynamic profile of phosphoproteome coupling bioinformatics, a complicated kinase-centered network related to BR-regulated growth was deciphered. The phosphoproteins and phosphosites identified from our study provide a useful dataset for revealing signaling networks of BR regulation, and also expanded our knowledge of protein phosphorylation modification in plants as well as further deal to solve the plant growth problems.

Dataset Information

Phosphoproteomics sample preparation impacts biological interpretation of signalling responses

Publications

Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes.

Similar Datasets

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