Affinity-based profiling of endogenous phosphoprotein phosphatases by mass spectrometry

Brauer, Brooke L; Wiredu, Kwame; Mitchell, Sierra; Moorhead, Greg B. G.; Gerber, Scott A.; Kettenbach, Arminja N.

doi:10.1038/s41596-021-00604-3

Cited by 7 publications

(16 citation statements)

References 67 publications

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“…Protein quantification was carried out by summing PSM areas on a per protein basis. Protein identifications were filtered to include known PPP subunits based on a curated list of previously identified subunits . Each subunit had to be identified with two or more peptides and be identified and quantified in all replicates of both cell lines (termed “all present”).…”

Section: Resultsmentioning

confidence: 99%

“…Data filtering and quantification were carried out as described above, requiring each PPP to be identified and quantified with more than one peptide in at least two replicates of a specific cell line. We limited this analysis to known PPP subunits based on a curated list of subunits we identified previously . Missing values were imputed, and proteins were normalized by quantile normalization.…”

Section: Resultsmentioning

confidence: 99%

“…PIBs were a gift from Greg Moorhead (University of Calgary) and can be synthesized according to ref . Cells were lysed in a high salt buffer [(50 mm Tris–HCl, pH = 8.0, Fisher Scientific), 500 mm NaCl (Fisher Scientific), 5 mm beta-glycerophosphate (Acros Organics), 0.5% Triton X-100 (Sigma), and 1:500 protease inhibitor cocktail III (Research Products International)], sonicated, clarified, and protein amount was quantified by the BCA assay (ThermoScientific).…”

Section: Methodsmentioning

confidence: 99%

“…Thus, subunit abundance often does not correlate with holoenzyme function, and investigations into PPP signaling require holoenzyme complex enrichment. To enable these inquiries, we developed a chemical proteomics strategy called phosphatase inhibitor beads and mass spectrometry (PIB-MS) . By employing an immobilized non-selective PPP inhibitor microcystin-LR, we can enrich the PPP subproteome (the “PPPome”) consisting of endogenous PP1, PP2A, PP4, PP5, and PP6 catalytic subunits and their interacting proteins (including scaffolding and regulatory subunits) from any cell or tissue type.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of Quantification and Normalization Strategies for Phosphoprotein Phosphatase Affinity Proteomics: Application to Breast Cancer Signaling

et al. 2022

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Accurate quantification of proteomics data is essential for revealing and understanding biological signaling processes. We have recently developed a chemical proteomic strategy termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) to investigate endogenous phosphoprotein phosphatase (PPP) dephosphorylation signaling. Here, we compare the robustness and reproducibility of status quo quantification methods for optimal performance and ease of implementation. We then apply PIB-MS to an array of breast cancer cell lines to determine differences in PPP signaling between subtypes. Breast cancer, a leading cause of cancer death in women, consists of three main subtypes: estrogen receptor-positive (ER+), human epidermal growth factor receptor two positive (HER2+), and triple-negative (TNBC). Although there are effective treatment strategies for ER+ and HER2+ subtypes, tumors become resistant and progress. Furthermore, TNBC has few targeted therapies. Therefore, there is a need to identify new approaches for treating breast cancers. Using PIB-MS, we distinguished TNBC from non-TNBC based on subtype-specific PPP holoenzyme composition. In addition, we identified an increase in PPP interactions with Hippo pathway proteins in TNBC. These interactions suggest that phosphatases in TNBC play an inhibitory role on the Hippo pathway and correlate with increased expression of YAP/TAZ target genes both in TNBC cell lines and in TNBC patients.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of Quantification and Normalization Strategies for Phosphoprotein Phosphatase Affinity Proteomics: Application to Breast Cancer Signaling

et al. 2022

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“…In this context, photoaffinity labeling (PAL) provides a powerful strategy for light-controlled protein modification in chemical proteomics. A popular application of PAL is coupling it with an affinity ligand for quantitative proteomics, commonly termed affinity-based protein profiling (A f BPP) . This methodology uses probes containing a certain photo-cross-linker to irreversibly link the molecule of interest to its target protein by photochemical reactions, thereby facilitating the target identification of bioactive molecules .…”

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confidence: 99%

Light-Controlled Traceless Protein Labeling via Decaging Thio-o-naphthoquinone Methide Chemistry

Wang

Huang

et al. 2022

Org. Lett.

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We report the molecular design of a novel multifunctional reagent and its application for light-controlled selective protein labeling. This molecule integrates functions of protein–ligand recognition, bioconjugation, ligand cleavage, and photoactivation by merging the photochemistries of 2-nitrophenylpropyloxycarbonyl and 3-hydroxymethyl-2-naphthol with an affinity ligand and fluorescein. Highly electrophilic o-naphthoquinone methide was photochemically released and underwent proximity-driven selective labeling with the protein of interest (e.g., carbonic anhydrases), which retains its native function after labeling.

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Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase

Egbert,

Silbern,

Uliasz

et al. 2023

Biology of Reproduction

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In response to luteinizing hormone, multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-minute exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the PPP family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4–10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles.

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Affinity-based profiling of endogenous phosphoprotein phosphatases by mass spectrometry

Cited by 7 publications

References 67 publications

Evaluation of Quantification and Normalization Strategies for Phosphoprotein Phosphatase Affinity Proteomics: Application to Breast Cancer Signaling

Evaluation of Quantification and Normalization Strategies for Phosphoprotein Phosphatase Affinity Proteomics: Application to Breast Cancer Signaling

Light-Controlled Traceless Protein Labeling via Decaging Thio-o-naphthoquinone Methide Chemistry

Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase

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