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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Abstract

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Introduction

Protein phosphorylation controls most cellular processes, including but not limited to the response to DNA damage, growth factor signaling, and the passage through mitosis1,2,3. In mammalian cells, the majority of proteins are phosphorylated at one or more serine, threonine, or tyrosine residues at some point in time, with phosphoserines and phosphothreonines comprising approximately 98% of all phosphorylation sites2,3. While kinases have been extensively studied in cellular signaling, the role of PPPs in the regulati....

Protocol

NOTE: The generation of PIBs is done as described by Moorhead et al., where 1 mg of microcystin and about 6 mL of sepharose are coupled to generate PIBs with a binding capacity of up to 5 mg/mL17.

1. Sample preparation

NOTE: A typical starting amount for PIB-MS is 1 mg of total protein per condition. For this experiment, approximately 2.5 x 106 HeLa cells were used to extract 1 mg of protein. This calculation should .......

Representative Results

figure-representative results-90
Figure 2: Identification of specific PIBs binders. (A) A variety of tissue types or cells can be analyzed via PIB-MS. HeLa cells in biological triplicate were either treated with DMSO or the PPP-inhibitor MCLR, incubated with PIBs, and analyzed via LC-MS/MS. (B) Volcano plot of PIB-MS anal.......

Discussion

PIB-MS is a chemical proteomics approach used to quantitatively profile the PPPome from various sample sources in a single analysis. Much work has been done using kinase inhibitor beads to study the kinome and how it changes in cancer and other disease states10,11,12,13. Yet, the study of the PPPome lags behind. We anticipate that this approach is able to fill this gap and shed light on the reg.......

Acknowledgements

A.N.K. acknowledges support from NIH R33 CA225458 and R35 GM119455. We thank the Kettenbach and Gerber labs for their helpful discussion.

....

Materials

NameCompanyCatalog NumberComments
Acetonitrile (ACN)HoneywellAH015-4CAUTION: ACN is flammable and toxic; wear gloves, and work in a chemical fume hood.
Anhydrous AcetonitrileSigma-Aldrich271004-100MLCAUTION: ACN is flammable and toxic; wear gloves, and work in a chemical fume hood.
Benchtop centrifugeEppendorfmodel no. 5424
Beta-glycerophosphoric acid, disodium salt pentahydrateAcros Organics410991000
CentrifugeEppendorfmodel no. 5810 R 15 amp version
Distilled water
DMSOFisher ScientificBP231-100
Dounce tissue grinderFisherbrand Pellet Pestles12-141-363
Empore solid phase extraction disk, C18CDS Analytical76333-132
Eppendorf tubes, 1.5 mLEppendorf22363204CRITICAL: Other tubes may leach polymer into sample, contaminating the analysis.
Eppendorf tubes, 2 mLEppendorf22363352CRITICAL: Other tubes may leach polymer into sample, contaminating the analysis.
Extraction plate manifoldWatersWAT097944
Falcon tubes, 50 mLVWR21008
Generic blunt end needle and plunger
Generic magnetic separation rack
HEPESSigma-AldrichH3375
Hydrogen chloride (HCl)VWR Chemicals BDHBDH3028CAUTION: HCl is corrosive; wear gloves and work in a chemical fume hood.
Hydroxylamine solution 50% (wt/vol)Sigma-Aldrich467804
Incubator, 65 °CVWRmodel no. 1380FM
Koptec Pure Ethanol, 200 ProofDecon LabsV1001
Methanol for HPLC (MeOH)Sigma-Aldrich34860-4L-RCAUTION: MeOH is flammable and toxic; wear gloves, and work in a chemical fume hood.
Microcystin LR (MCLR)Cayman Chemical10007188CAUTION: MCLR is toxic; wear gloves when handling and avoid skin contact.
PBS, 1× without calcium and magnesium, pH 7.4 ± 0.1Corning 21-040-CV
pH test strips, such as MilliporeSigma MColorpHast pH test strips and indicator papersFisher ScientificM1095310001
PIBsFor protocol for the generation of PIBs, see Moorhead et al., 2007.
Pierce BCA Protein Assay KitThermo Scientific23225
Pipette tips, 10 μLEppendorf22491504CRITICAL: Other tips may leach polymer into samples, contaminating the analysis.
Pipette tips, 1000 μLEppendorf22491555CRITICAL: Other tips may leach polymer into samples, contaminating the analysis.
Pipette tips, 200 μLEppendorf22491539CRITICAL: Other tips may leach polymer into samples, contaminating the analysis.
plastic syringe, 10 mLBD309604
Protease inhibitor cocktail IIIResearch Products InternationalP50700-1
Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer, Oribtrap Fusion, Orbitrap Fusion Lumos, or Orbitrap Eclipse Tribrid Mass Spectrometer Thermo Scientific
Refrigerated benchtop centrifugeEppendorfmodel no. 5424 R
Rotator (Labquake Shaker Rotisserie)Thermo Scientific13-687-12Q8 rpm rotation
Sample collection plate, 96- well, 1 mLWatersWAT058957
SDSFisher ScientificBP1311-1
Sequencing grade modified trypsinPromegaV511C
Sodium azideEMD ChemicalsSX0299-1CAUTION: Sodium azide is explosive and toxic; wear gloves, work in a chemical fume hood and avoid contact with metals.
Sodium chloride (NaCl)Fisher ChemicalS27110
Sonicator (Branson digital sonifier)model no. SFX 250
SPE C18 desalting plateWaters186001828BA
SpeedBeads magnetic carboxylate modified particles (SP3 beads)Cytiva6.51521E+13
ThermomixerEppendorfmodel no. 5350
TMT10plex Isobaric Label Reagent Set plus TMT11-131C Label Reagent, 3 × 0.8 mg per tagThermoFisherA37725
Trifluoroacetic acid (TFA)HoneywellT6508-25MLCAUTION: TFA is corrosive and will irritate skin on contact. Wear gloves and eye protection, and work in a chemical fume hood.
Tris BaseResearch Products InternationalT60040
Triton X-100Sigma-AldrichT9284
Vacuum centrifuge and vapor trapThermo Scientificmodel nos. SpeedVac SPD120 and RVT5105
Vortexer (Vortex-Genie 2)Scientific Industries
Water LC-MSHoneywellLC365-4

References

  1. Nilsson, J. Protein phosphatases in the regulation of mitosis. Journal of Cell Biology. 218 (2), 395-409 (2019).
  2. Brautigan, D. L. Protein Ser/Thr phosphatases--the ugly ducklings of cell signalling. T....

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