Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca2+-responsive pathways
- Whitehead Institute for Biomedical Research, United States
- Biology Department, Massachusetts Institute of Technology, United States
- Center for Tropical and Emerging Global Diseases, University of Georgia, United States
Figures
Figure 1 with 1 supplement
Phosphoregulation triggered by Ca2+ release.
(A) Schematic of the sub-minute phosphoproteomics experiments with the Ca2+ signaling agonist zaprinast. (B) The summed abundances of unique phosphopeptides during zaprinast or vehicle (DMSO) …
Figure 1—figure supplement 1
Metrics describing the zaprinast-dependent phosphoproteome.
(A) Aggregate protein abundances for all time points from the non-phosphopeptide enriched samples of parasites treated with zaprinast or the corresponding vehicle (DMSO). Proteins quantified by a …
Figure 2 with 1 supplement
Thermal profiling identifies proteins that change stability in response to Ca2+.
(A) Thermal shift assays can detect Ca2+-dependent stability of CDPK1 in extracts. Parasite lysates were combined with 10 concentrations of Ca2+ spanning the nanomolar to micromolar range. After …
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Figure 2—source data 1
This file contains the source data that was quantified to make the graph presented in Figure 2.
TUB1, LICOR 700 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig2-data1-v3.zip
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Figure 2—source data 2
This file contains the source data that was quantified to make the graph presented in Figure 2.
CDPK1, LICOR 800 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig2-data2-v3.zip
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Figure 2—source data 3
This file contains the annotated source data that was quantified to make the graph presented in Figure 2.
TUB1, LICOR 700 channel and CDPK1, LICOR 800 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig2-data3-v3.zip
Figure 2—figure supplement 1
Extended data for thermal profiling experiments.
(A) Comparison of the melting points of proteins with standard melting behavior (R2 >0.8) across replicate experiments and treatment conditions. The correlation of melting temperatures is given by …
Figure 3 with 1 supplement
Thermal profiling identifies anticipated and unexplored Ca2+-responsive proteins.
(A) Mass spectrometry-derived thermal profiles of EF hand-containing proteins stabilized or destabilized by Ca2+. Relative abundance is calculated relative to the protein abundance at 0 µM Ca2+. EC50…
Figure 3—figure supplement 1
Extended analysis of thermal profiling experiments.
(A) Plots of protein curve fit R2 vs. AUC, a measure of stability change, for each set of MS experiments. Dotted lines indicate thresholds for designated Ca2+-responsive behavior: R2 >0.8 and an AUC …
Figure 4 with 1 supplement
Validation of Ca2+-dependent thermal stability.
(A) Mass spectrometry-derived thermal profiles of the candidates, as in Figure 3A. (B) Immunofluorescence images of fixed intracellular parasites expressing the indicated proteins with C-terminal …
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Figure 4—source data 1
This file contains the source data that was quantified to make the graph presented in Figure 4.
TUB1, LICOR 700 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig4-data1-v3.zip
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Figure 4—source data 2
This file contains the source data that was quantified to make the graph presented in Figure 4.
PKA C1-Ty, LICOR 800 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig4-data2-v3.zip
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Figure 4—source data 3
This file contains the source data that was quantified to make the graph presented in Figure 4.
TUB1, LICOR 700 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig4-data3-v3.zip
-
Figure 4—source data 4
This file contains the source data that was quantified to make the graph presented in Figure 4.
Eps15-HA LICOR 800 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig4-data4-v3.zip
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Figure 4—source data 5
This file contains the source data that was quantified to make the graph presented in Figure 4.
286710 HA, LICOR 700 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig4-data5-v3.zip
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Figure 4—source data 6
This file contains the source data that was quantified to make the graph presented in Figure 4.
MIC2, LICOR 800 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig4-data6-v3.zip
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Figure 4—source data 7
This file contains the source data that was quantified to make the graph presented in Figure 4.
GAP45, LICOR 700 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig4-data7-v3.zip
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Figure 4—source data 8
This file contains the source data that was quantified to make the graph presented in Figure 4.
309290-V5, LICOR 800 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig4-data8-v3.zip
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Figure 4—source data 9
This file contains the source data that was quantified to make the graph presented in Figure 4.
SAG1, LICOR 700 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig4-data9-v3.zip
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Figure 4—source data 10
This file contains the source data that was quantified to make the graph presented in Figure 4.
RON13-HA, LICOR 800 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig4-data10-v3.zip
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Figure 4—source data 11
This file contains the annotated source data that was quantified to make the graph presented in Figure 4.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig4-data11-v3.zip
Figure 4—figure supplement 1
Uncropped immunoblots, as in Figure 4C.
Parasite lysates were incubated at 10 Ca2+ concentrations and were thermally challenged at 58 °C. Following centrifugation, the supernatant containing the soluble protein fraction was separated by …
Figure 5
PP1 is a Ca2+-responsive enzyme involved in T. gondii egress and invasion.
(A) The Ca2+-dependent stabilization of PP1 (TGGT1_310700) in each mass spectrometry experiment. (B) Immunoblotting for endogenously tagged PP1-mNG-Ty at different Ca2+ concentrations and thermal …
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Figure 5—source data 1
This file contains the source data that was quantified to make the graph presented in Figure 4.
ALD1, LICOR 700 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig5-data1-v3.zip
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Figure 5—source data 2
This file contains the source data that was quantified to make the graph presented in Figure 4.
PP1-Ty, LICOR 800 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig5-data2-v3.zip
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Figure 5—source data 3
This file contains the annotated source data that was quantified to make the graph presented in Figure 5.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig5-data3-v3.zip
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Figure 5—source data 4
This file contains the source data that was presented in Figure 5.
CDPK1, LICOR 700 channel (magenta) and PP1-HA, LICOR 800 channel (green).
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig5-data4-v3.zip
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Figure 5—source data 5
This file contains the annotate source data that was presented in Figure 5.
CDPK1, LICOR 700 channel (magenta) and PP1-HA, LICOR 800 channel (green).
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig5-data5-v3.zip
Figure 6 with 1 supplement
The PP1-dependent phosphoproteome.
(A) Schematic of the phosphoproteomics time course. PP1-AID parasites were treated with IAA or vehicle for 3 hr. Extracellular parasites were then treated with zaprinast, and samples were collected …
Figure 6—figure supplement 1
Extended analysis of PP1 phosphoproteomics experiment.
(A) Aggregate protein abundances from the non-phosphopeptide enriched samples of parasites treated with IAA or vehicle. Proteins quantified by a single peptide or more are shown in light and dark …
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Figure 6—figure supplement 1—source data 1
This file contains the source data that was presented in Figure 6—figure supplement 1.
CDPK1, LICOR 700 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig6-figsupp1-data1-v3.zip
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Figure 6—figure supplement 1—source data 2
This file contains the source data that was presented in Figure 6—figure supplement 1.
PP1-HA, LICOR 800 channel.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig6-figsupp1-data2-v3.zip
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Figure 6—figure supplement 1—source data 3
This file contains the annotated source data that was presented in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/80336/elife-80336-fig6-figsupp1-data3-v3.zip
Figure 7 with 1 supplement
PP1 ensures rapid Ca2+ mobilization prior to zaprinast-induced egress.
(A) Selected frames of time-lapse images of PP1-AID parasites expressing the genetically encoded Ca2+ indicator GCaMP following treatment with 500 µM zaprinast. (B) Normalized GCaMP fluorescence of …
Figure 7—figure supplement 1
PP1-AID parasites egress, as quantified by video microscopy.
(A) The time to vacuole egress after zaprinast treatment was manually scored. Different replicates are shown in different shades of gray. Small points represent individual vacuoles; large points are …
Videos
Video 1
Representative image series of parasites expressing endogenously tagged PP1-mNG following treatment with 500 µM zaprinast.
Video 2
Representative image series of parasites expressing endogenously tagged PP1-mNG following treatment with 4 µM A23187.
Video 3
Representative image series of PP1-AID parasites expressing the genetically encoded Ca2+ indicator GCaMP following treatment with 500 µM zaprinast.
Video 4
Representative image series of PP1-AID parasites expressing the genetically encoded Ca2+ indicator GCaMP following treatment with 4 µM A23187.
Tables
Table 1
Gene IDs of proteins discussed in the text.
NS, not significant (using thresholds defined in the text). ND, not detected. TR, temperature range. CR, concentration range.
| Gene ID | Description in text | Reference | Phospho cluster | Thermal profiling |
|---|---|---|---|---|
| TGGT1_202540 | PDE1 | Jia et al., 2017 | 1, 3 | NS |
| TGGT1_293000 | PDE2 | Jia et al., 2017; Moss et al., 2022; Vo et al., 2020 | 1, 2, 4 | NS |
| TGGT1_245730 | PI4,5K | Garcia et al., 2017 | 1, 2 | NS |
| TGGT1_276170 | PI3,4K | Garcia et al., 2017 | 1, 2 | NS |
| TGGT1_248830 | PI-PLC | Bullen et al., 2016; Fang et al., 2006 | 1, 3 | NS |
| TGGT1_288800 | Phosphatidylinositol-3,4,5-triphosphate 5-phosphatase | 1 | NS | |
| TGGT1_254390 | Putative Sec14 | 1 | NS | |
| TGGT1_206590 | CDPK2A | Billker et al., 2009 | 1, 2, 3 | CR |
| TGGT1_228750 | CDPK7 | Bansal et al., 2021 | 1, 3 | TR |
| TGGT1_267100 | PPM2B | Yang et al., 2019; Yang and Arrizabalaga, 2017 | 1 | NS |
| TGGT1_238995 | Ca2+-activated K+ channel | 2 | ND | |
| TGGT1_273380 | Ca2+-activated K+ channel | 2, 3 | ND | |
| TGGT1_259200B | Na+/H+ exchanger | Arrizabalaga et al., 2004 | 2, 3 | NS |
| TGGT1_305180 | Na+/H+ exchanger | Francia et al., 2011 | 2, 3, 4 | NS |
| TGGT1_245510 | ATPase2 | Chen et al., 2021 | 2 | ND |
| TGGT1_254370 | Guanylyl cyclase | Bisio et al., 2019; Brown and Sibley, 2018 | 1, 2, 3, 4 | NS |
| TGGT1_312100 | Calcium ATPase TgA1 | Luo et al., 2005; Luo et al., 2001 | 3 | ND |
| TGGT1_201150 | Copper transporter CuTP | Kenthirapalan et al., 2014 | 3 | NS |
| TGGT1_318460 | Putative P5B-ATPase | Møller et al., 2008 | 2 | NS |
| TGGT1_289070 | Putative E1-E2 ATPase | 3 | NS | |
| TGGT1_278660 | TgATP4 | Lehane et al., 2019 | 3, 4 | NS |
| TGGT1_226020 | MFS transporter | 3 | NS | |
| TGGT1_230570 | MFS transporter | 3 | NS | |
| TGGT1_253700 | MFS transporter | 3, 4 | NS | |
| TGGT1_257530 | Tyrosine transporter ApiAT5-3 | Parker et al., 2019; Wallbank et al., 2019 | 3, 4 | NS |
| TGGT1_292110 | Formate transporter TgFNT2 | Erler et al., 2018; Zeng et al., 2021 | 3 | NS |
| TGGT1_270865 | Adenylyl cyclase | Brown and Sibley, 2018; Jia et al., 2017 | 2, 3 | NS |
| TGGT1_238390 | Unique guanylyl cyclase organizer UGO | Bisio et al., 2019 | 2, 3 | NS |
| TGGT1_309190 | ARO-interacting protein (adenylyl cyclase organizer) AIP | Mueller et al., 2016; Mueller et al., 2013 | 1, 3 | NS |
| TGGT1_273560 | Divergent kinesin KinesinB | Leung et al., 2017 | 2, 3 | NS |
| TGGT1_201230 | Divergent kinesin | Wickstead et al., 2010 | 3 | ND |
| TGGT1_247600 | Dynein light chain | 3 | NS | |
| TGGT1_255190 | MyoC | Frénal et al., 2017 | 3 | NS |
| TGGT1_278870 | MyoF | Heaslip et al., 2016; Jacot et al., 2013 | 1, 2, 3 | NS |
| TGGT1_257470 | MyoJ | Frénal et al., 2014 | 3 | CR |
| TGGT1_213325 | Uncharacterized TBC domain protein | 3 | ND | |
| TGGT1_221710 | Uncharacterized TBC domain protein | 3 | NS | |
| TGGT1_237280 | Uncharacterized TBC domain protein | 1, 3, 4 | NS | |
| TGGT1_274130 | Uncharacterized TBC domain protein | 2, 3, 4 | NS | |
| TGGT1_289820 | Uncharacterized TBC domain protein | 3 | NS | |
| TGGT1_206690 | GAPM2B | Harding et al., 2019 | 3 | NS |
| TGGT1_271970 | GAPM3 | Harding et al., 2019 | 3 | NS |
| TGGT1_233010 | ERK7 | O’Shaughnessy et al., 2020 | 3, 4 | CR |
| TGGT1_234970 | Tyrosine kinase-like protein TgTLK2 | Varberg et al., 2018 | 4 | NS |
| TGGT1_202900 | Putative K+ voltage-gated channel complex subunit | 4 | NS | |
| TGGT1_228200 | Vacuolar (H+)-ATPase G subunit | 1, 4 | NS | |
| TGGT1_233130 | Putative nucleoside transporter | 4 | NS | |
| TGGT1_258700 | MFS family transporter | 4 | ND | |
| TGGT1_269260 | SCE1 | McCoy et al., 2017 | 1, 2, 3 | NS |
| TGGT1_295850 | AAP2 | Engelberg et al., 2020 | 1, 2, 4 | CR |
| TGGT1_319900 | AAP5 | Engelberg et al., 2020 | 1, 2, 3, 4 | NS |
| TGGT1_227000 | Apical polar ring protein | Koreny et al., 2021 | 1, 3, 4 | ND |
| TGGT1_244470 | RNG2 | Katris et al., 2014 | 1, 2, 3 | ND |
| TGGT1_292950 | Apical cap protein AC10 | Back et al., 2020; Tosetti et al., 2020 | 1, 2, 3 | NS |
| TGGT1_240380 | Conoid gliding protein CGP | Li et al., 2022 | 1, 3, 4 | NS |
| TGGT1_216620 | Ca2+ influx channel with EF hands | Chang et al., 2019 | 1, 3, 4 | NS |
| TGGT1_246930 | Calmodulin-like protein CAM1 | Long et al., 2017b | ND | TR |
| TGGT1_262010 | ACalmodulin-like protein CAM2 | Long et al., 2017b | ND | TR, CR |
| TGGT1_216080 | apical lysine methyltransferase (AKMT) | Heaslip et al., 2011 | NS | TR |
| TGGT1_270690 | DrpC | Heredero-Bermejo et al., 2019; Melatti et al., 2019 | NS | TR |
| TGGT1_201880 | TgQCR9 | ND | TR | |
| TGGT1_204400 | ATP synthase subunit alpha | Hayward et al., 2021; Huet et al., 2018; Mühleip et al., 2021; Salunke et al., 2018; Seidi et al., 2018 | NS | TR |
| TGGT1_231910 | ATP synthase subunit gamma | Huet et al., 2018; Mühleip et al., 2021; Salunke et al., 2018; Seidi et al., 2018 | ND | TR |
| TGGT1_208440 | ATP synthase subunit 8/ASAP-15 | Huet et al., 2018; Mühleip et al., 2021; Salunke et al., 2018; Seidi et al., 2018 | ND | TR, CR |
| TGGT1_215610 | ATP synthase subunit f/ICAP11/ASAP-10 | Huet et al., 2018; Mühleip et al., 2021; Salunke et al., 2018; Seidi et al., 2018 | ND | TR |
| TGGT1_263080 | ATP synthase-associated protein ASAP-18/ATPTG14 | Huet et al., 2018; Mühleip et al., 2021; Salunke et al., 2018; Seidi et al., 2018 | ND | TR, CR |
| TGGT1_246540 | ATP synthase-associated protein ATPTG1 | Mühleip et al., 2021 | NS | TR |
| TGGT1_249240 | CaM | Paul et al., 2015 | ND | CR |
| TGGT1_213800 | CnB | Paul et al., 2015 | NS | CR |
| TGGT1_227800 | Eps15 | Birnbaum et al., 2020; Chern et al., 2021 | NS | CR |
| TGGT1_269442 | ELC1 | Nebl et al., 2011 | ND | CR |
| TGGT1_297470 | MLC1 | Gaskins et al., 2004 | ND | CR |
| TGGT1_297470 | MLC5 | Graindorge et al., 2016 | ND | CR |
| TGGT1_315780 | MLC7 | Graindorge et al., 2016 | ND | CR |
| TGGT1_226030 | PKA-C1 | Jia et al., 2017; Uboldi et al., 2018 | NS | CR |
| TGGT1_242070 | PKA-R | Jia et al., 2017; Uboldi et al., 2018 | NS | CR |
| TGGT1_210830 | Putative RIO1 kinase | NS | CR | |
| TGGT1_310700 | PP1 | Paul et al., 2020; Zeeshan et al., 2021 | ND | CR |
| TGGT1_207910 | Calcium-hydrogen exchanger TgCAX | Guttery et al., 2013 | NS | CR |
| TGGT1_311080 | Apicoplast two-pore channel TgTPC | Li et al., 2021 | NS | CR |
| TGGT1_204050 | Subtilisin 1 SUB1 | NS | CR | |
| TGGT1_206490 | Metacaspase 1 with a C2 domain | Li et al., 2015 | ND | CR |
| TGGT1_310810 | Ca2+-activated apyrase | ND | CR | |
| TGGT1_321650 | RON13 | Lentini et al., 2021 | ND | CR |
| TGGT1_286710 | Uncharacterized metal-binding protein with zinc fingers | ND | CR | |
| TGGT1_309290 | Uncharacterized metal-binding protein with HD domain | ND | CR | |
| TGGT1_225690 | Apical cap protein AC7 | Chen et al., 2015 | 2, 3 | NS |
| TGGT1_234250 | CHP interacting protein CIP1 | Long et al., 2017a | NS | ND |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (T. gondii) | TIR1 | PMID:28465425 | RH/TIR1/∆KU80/∆HXGPRT | |
| Strain, strain background (T. gondii) | TIR1/GCaMP6f | PMID:35484233 | RH/TIR1/pTUB1-GCaMP6f-3'DHFR/∆KU80/∆HXGPRT | |
| Strain, strain background (T. gondii) | DiCre | PMID:31577230 | RH/∆KU80::DiCre_T2A/∆HXGPRT | |
| Strain, strain background (T. gondii) | TIR1/MIC2-GLuc-P2A-GCaMP6f | This paper | RH/TIR1/∆KU80/∆HXGPRT/pMIC2-MIC2-GLuc-myc-P2A-GCaMP6f | |
| Strain, strain background (T. gondii) | PKA C1-Ty (Figure 4) | This paper | TGGT1_226030 | RH/TIR1/pTUB1-GCaMP6f-3'DHFR/∆KU80/PKA C1-mCherry-V5-mAID-Ty/PKA R-V5-3HA |
| Strain, strain background (T. gondii) | Eps15-HA (Figure 4) | This paper | TGGT1_227800 | RH/TIR1/pTUB1-GCaMP6f-3'DHFR/∆KU80/Eps15-V5-mCherry-mAID-HA |
| Strain, strain background (T. gondii) | zinc finger-HA (Figure 4) | This paper | TGGT1_286710 | RH/TIR1/∆KU80/∆HXGPRT/TGGT1_286470-V5-3HA |
| Strain, strain background (T. gondii) | hypothetical-V5 (Figure 4) | This paper | TGGT1_309290 | RH/TIR1/pTUB1-GCaMP6f-3'DHFR/∆KU80/∆HXGPRT/TGGT1_309290-V5-mNeonGreen-mAID-Ty |
| Strain, strain background (T. gondii) | RON13-HA (Figure 4) | This paper | TGGT1_321650 | RH/∆KU80::DiCre_T2A/∆HXGPRT/RON13-HA-U1 |
| Strain, strain background (T. gondii) | PP1-AID (Figure 5) | This paper | TGGT1_310700 | RH/TIR1/∆KU80/∆HXGPRT/pMIC2-MIC2-GLuc-myc-P2A-GCaMP6f/PP1-V5-mAID-HA |
| Strain, strain background (T. gondii) | PP1-AID/TIR1/MIC2-GLuc-P2A-GCaMP6f (Figure 7) | This paper | TGGT1_310700 | RH/TIR1/∆KU80/∆HXGPRT/pMIC2-MIC2-GLuc-myc-P2A-GCaMP6f/PP1-V5-mAID-HA |
| Cell line (Homo sapiens) | Human Foreskin Fibroblasts (HFFs) | ATCC | SCRC-1041 | |
| Antibody | Guinea pig monoclonal anti-CDPK1 | Covance | Custom antibody | IF (1/10000), WB (1/40000) |
| Antibody | Mouse monoclonal anti-TUB1 (clone 12G10) | Developmental Studies Hybridoma Bank at the University of Iowa | RRID:AB_1157911 | WB (1/5000) |
| Antibody | Mouse monoclonal anti-Ty1 (clone BB2) | PMID:8813669 | IF (1/1000), WB(1/2000) | |
| Antibody | Rabbit polyclonal anti-HA | Invitrogen | Invitrogen:71–5500 | WB (1/1000) |
| Antibody | Mouse monoclonal anti-MIC2 (6D10) | PMID:10799515 | WB (1:2000) | |
| Antibody | Mouse monoclonal anti-V5 | Invitrogen | Invitrogen:R960-25 | IF (1:1000), WB (1:2000) |
| Antibody | Rabbit polyclonal | PMID:18312842 | WB (1:2000) | |
| Antibody | Mouse monoclonal anti-HA (16B12) | Biolegend | Biolegend:901533 | IF (1:1000) |
| Antibody | Mouse polyclonal anti-SAG1 | PMID:3183382 | WB (1/1000) | |
| Antibody | Alexa Fluor 594 polyclonal goat anti-guinea pig | Life Technologies | Life Technologies:A11076 | IF (1/1000) |
| Antibody | Alexa Fluor 488 polyclonal goat anti-mouse | Life Technologies | Life Technologies:A11029 | IF (1/1000) |
| Antibody | IRDye 800CW polyclonal Goat anti-Mouse IgG1-Specific Secondary Antibody | LICOR | LICOR:926–32350 | WB (1/10000) |
| Antibody | IRDye 680LT polyclonal Goat anti-Mouse IgG Secondary Antibody | LICOR | LICOR:926–68020 | WB (1/10000) |
| Antibody | IRDye 800CW polyclonal Donkey anti-Guinea Pig IgG Secondary Antibody | LICOR | LICOR:926–32411 | WB (1/10000) |
| Antibody | IRDye 680RD polyclonal Donkey anti-Guinea Pig IgG Secondary Antibody | LICOR | LICOR:926–68077 | WB (1/10000) |
| Antibody | IRDye 800CW polyclonal Goat anti-Rabbit IgG Secondary Antibody | LICOR | LICOR:926–32211 | WB (1/10000) |
| Antibody | IRDye 680LT polyclonal Goat anti-Rabbit IgG Secondary Antibody | LICOR | LICOR:926–68021 | WB (1/10000) |
| Chemical compound, drug | Hoechst | Santa Cruz | Santa Cruz:sc-394039 | IF (1/20000) |
| Chemical compound, drug | Prolong Diamond | Thermo Fisher | Thermo Fisher:P36965 | |
| Chemical compound, drug | zaprinast | Calbiochem | Calbiochem:684500 | |
| Chemical compound, drug | A23187 | Calbiochem | Calbiochem:100105 | |
| Commercial assay or kit | S-trap micro | Protifi | Protific:C02-micro-80 | |
| Commercial assay or kit | TMT10plex Isobaric Label Reagent Set | Thermo Fisher Scientific | Thermo Fisher Scientific:90111 | |
| Commercial assay or kit | TMTpro 16plex Label Reagent Set | Thermo Fisher Scientific | Thermo Fisher Scientific:A44522 | |
| Commercial assay or kit | High-Select TiO2 Phosphopeptide Enrichment Kit | Thermo Fisher Scientific | Thermo Fisher Scientific:A32993 | |
| Commercial assay or kit | High-Select Fe-NTA Phosphopeptide Enrichment Kit | Thermo Fisher Scientific | Thermo Fisher Scientific:A32992 | |
| Commercial assay or kit | Pierce High pH Reversed-Phase Peptide Fractionation Kit | Thermo Fisher Scientific | Thermo Fisher Scientific:84868 | |
| Commercial assay or kit | Calcium Calibration Buffer Kit #1, zero and 10 mM CaEGTA | Life Technologies | Life Technologies:C3008MP | |
| Commercial assay or kit | Hydrophobic Sera-Mag Speed Beads | GE Healthcare | GE Healthcare:65152105050250 | |
| Commercial assay or kit | Hydrophilic Sera-Mag Speed Beads | GE Healthcare | GE Healthcare:45152105050250 | |
| Recombinant DNA reagent | All plasmids used in this study are listed in Supplementary file 7 | |||
| Sequence-based reagent | All primers and oligonucleotides used in this study are listed in Supplementary file 7 | |||
| Software, algorithm | Proteome Discoverer 4.2 | Thermo Fisher | ||
| Software, algorithm | R version 4.0 | R Foundation for Statistical Computing | ||
| Software, algorithm | mineCETSA version 1.1.1 | Dziekan et al., 2020 | https://github.com/nkdailingyun/mineCETSA | |
| Software, algorithm | Prism 8 | GraphPad | ||
| Software, algorithm | HHPRED | PMID:29258817 | ||
| Other | Halt protease inhibitor | Thermo Fisher | Thermo Fisher:87786 | Materials and Methods: lysis buffer |
| Other | Halt protease and phosphatase inhibitor | Thermo Fisher | Thermo Fisher:PI78440 | Materials and Methods: lysis buffer |
| Other | Benzonase | Sigma Aldrich | Sigma Aldrich:E1014 | Materials and Methods: lysis buffer |
Additional files
-
Supplementary file 1
Sub-minute phosphoproteomics time course protein and abundance assignments from Proteome Discoverer 2.4.
- https://cdn.elifesciences.org/articles/80336/elife-80336-supp1-v3.txt
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Supplementary file 2
Sub-minute phosphoproteomics time course peptide and abundance assignments from Proteome Discoverer 2.4.
Mclust cluster assignments (column 118) of phosphopeptides dynamically changing during zaprinast treatment.
- https://cdn.elifesciences.org/articles/80336/elife-80336-supp2-v3.txt
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Supplementary file 3
Data pertaining to the temperature range thermal profiling experiment.
1. Protein and abundance assignments from Proteome Discoverer 2.4 for samples with 0 µM Ca2+, replicate 1. 2. Protein and abundance assignments from Proteome Discoverer 2.4 for samples with 0 µM Ca2+, replicate 2. 3. Protein and abundance assignments from Proteome Discoverer 2.4 for samples with 10 µM Ca2+, replicate 1. 4. Protein and abundance assignments from Proteome Discoverer 2.4 for samples with 10 µM Ca2+, replicate 2. 5. Curve fit output from the mineCETSA package. 6. Area under the euclidean distance score calculations from the mineCETSA package.
- https://cdn.elifesciences.org/articles/80336/elife-80336-supp3-v3.xlsx
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Supplementary file 4
Data pertaining to the concentration range thermal profiling experiments.
1. Protein and abundance assignments from Proteome Discoverer 2.4 for Experiment 1 samples with 54 °C, replicate 1. 2. Protein and abundance assignments from Proteome Discoverer 2.4 for Experiment 1 samples with 54 °C, replicate 2. 3. Protein and abundance assignments from Proteome Discoverer 2.4 for Experiment 1 samples with 58 °C, replicate 1. 4. Protein and abundance assignments from Proteome Discoverer 2.4 for Experiment 1 samples with 58 °C, replicate 2. 5. Curve fit output for concentration range Experiment 1 from the mineCETSA package. 6. Area under the curve score calculations from the mineCETSA package for concentration range Experiment 2. 7. Protein and abundance assignments from Proteome Discoverer 2.4 for Experiment 2 samples with 50 °C, replicate 1. 8. Protein and abundance assignments from Proteome Discoverer 2.4 for Experiment 2 samples with 50 °C, replicate 2. 8. Protein and abundance assignments from Proteome Discoverer 2.4 for Experiment 2 samples with 54 °C, replicate 1. 9. Protein and abundance assignments from Proteome Discoverer 2.4 for Experiment 2 samples with 54 °C, replicate 2. 10. Protein and abundance assignments from Proteome Discoverer 2.4 for Experiment 2 samples with 58 °C, replicate 1. 11. Protein and abundance assignments from Proteome Discoverer 2.4 for Experiment 2 samples with 58 °C, replicate 2. 12. Curve fit output for concentration range Experiment 2 from the mineCETSA package. 13. Area under the curve score calculations from the mineCETSA package for concentration range Experiment 2.
- https://cdn.elifesciences.org/articles/80336/elife-80336-supp4-v3.xlsx
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Supplementary file 5
PP1 depletion zaprinast phosphoproteomics time course protein and abundance assignments from Proteome Discoverer 2.4.
- https://cdn.elifesciences.org/articles/80336/elife-80336-supp5-v3.txt
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Supplementary file 6
PP1 depletion zaprinast phosphoproteomics time course peptide and abundance assignments from Proteome Discoverer 2.4.
Mclust cluster assignments (column 2) of phosphopeptides dynamically changing during zaprinast treatment when PP1 is depleted.
- https://cdn.elifesciences.org/articles/80336/elife-80336-supp6-v3.txt
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Supplementary file 7
Sequences and accessions of oligonucleotides and plasmids used in this study.
- https://cdn.elifesciences.org/articles/80336/elife-80336-supp7-v3.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/80336/elife-80336-mdarchecklist1-v3.docx
