A subscription to JoVE is required to view this content. Sign in or start your free trial.
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.
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.
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....
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 .......
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.......
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.......
A.N.K. acknowledges support from NIH R33 CA225458 and R35 GM119455. We thank the Kettenbach and Gerber labs for their helpful discussion.
....Name | Company | Catalog Number | Comments |
Acetonitrile (ACN) | Honeywell | AH015-4 | CAUTION: ACN is flammable and toxic; wear gloves, and work in a chemical fume hood. |
Anhydrous Acetonitrile | Sigma-Aldrich | 271004-100ML | CAUTION: ACN is flammable and toxic; wear gloves, and work in a chemical fume hood. |
Benchtop centrifuge | Eppendorf | model no. 5424 | |
Beta-glycerophosphoric acid, disodium salt pentahydrate | Acros Organics | 410991000 | |
Centrifuge | Eppendorf | model no. 5810 R 15 amp version | |
Distilled water | |||
DMSO | Fisher Scientific | BP231-100 | |
Dounce tissue grinder | Fisherbrand Pellet Pestles | 12-141-363 | |
Empore solid phase extraction disk, C18 | CDS Analytical | 76333-132 | |
Eppendorf tubes, 1.5 mL | Eppendorf | 22363204 | CRITICAL: Other tubes may leach polymer into sample, contaminating the analysis. |
Eppendorf tubes, 2 mL | Eppendorf | 22363352 | CRITICAL: Other tubes may leach polymer into sample, contaminating the analysis. |
Extraction plate manifold | Waters | WAT097944 | |
Falcon tubes, 50 mL | VWR | 21008 | |
Generic blunt end needle and plunger | |||
Generic magnetic separation rack | |||
HEPES | Sigma-Aldrich | H3375 | |
Hydrogen chloride (HCl) | VWR Chemicals BDH | BDH3028 | CAUTION: HCl is corrosive; wear gloves and work in a chemical fume hood. |
Hydroxylamine solution 50% (wt/vol) | Sigma-Aldrich | 467804 | |
Incubator, 65 °C | VWR | model no. 1380FM | |
Koptec Pure Ethanol, 200 Proof | Decon Labs | V1001 | |
Methanol for HPLC (MeOH) | Sigma-Aldrich | 34860-4L-R | CAUTION: MeOH is flammable and toxic; wear gloves, and work in a chemical fume hood. |
Microcystin LR (MCLR) | Cayman Chemical | 10007188 | CAUTION: MCLR is toxic; wear gloves when handling and avoid skin contact. |
PBS, 1× without calcium and magnesium, pH 7.4 ± 0.1 | Corning |  21-040-CV | |
pH test strips, such as MilliporeSigma MColorpHast pH test strips and indicator papers | Fisher Scientific | M1095310001 | |
PIBs | For protocol for the generation of PIBs, see Moorhead et al., 2007. | ||
Pierce BCA Protein Assay Kit | Thermo Scientific | 23225 | |
Pipette tips, 10 μL | Eppendorf | 22491504 | CRITICAL: Other tips may leach polymer into samples, contaminating the analysis. |
Pipette tips, 1000 μL | Eppendorf | 22491555 | CRITICAL: Other tips may leach polymer into samples, contaminating the analysis. |
Pipette tips, 200 μL | Eppendorf | 22491539 | CRITICAL: Other tips may leach polymer into samples, contaminating the analysis. |
plastic syringe, 10 mL | BD | 309604 | |
Protease inhibitor cocktail III | Research Products International | P50700-1 | |
Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer, Oribtrap Fusion, Orbitrap Fusion Lumos, or Orbitrap Eclipse Tribrid Mass Spectrometer | Thermo Scientific | ||
Refrigerated benchtop centrifuge | Eppendorf | model no. 5424 R | |
Rotator (Labquake Shaker Rotisserie) | Thermo Scientific | 13-687-12Q | 8 rpm rotation |
Sample collection plate, 96- well, 1 mL | Waters | WAT058957 | |
SDS | Fisher Scientific | BP1311-1 | |
Sequencing grade modified trypsin | Promega | V511C | |
Sodium azide | EMD Chemicals | SX0299-1 | CAUTION: Sodium azide is explosive and toxic; wear gloves, work in a chemical fume hood and avoid contact with metals. |
Sodium chloride (NaCl) | Fisher Chemical | S27110 | |
Sonicator (Branson digital sonifier) | model no. SFX 250 | ||
SPE C18 desalting plate | Waters | 186001828BA | |
SpeedBeads magnetic carboxylate modified particles (SP3 beads) | Cytiva | 6.51521E+13 | |
Thermomixer | Eppendorf | model no. 5350 | |
TMT10plex Isobaric Label Reagent Set plus TMT11-131C Label Reagent, 3 × 0.8 mg per tag | ThermoFisher | A37725 | |
Trifluoroacetic acid (TFA) | Honeywell | T6508-25ML | CAUTION: TFA is corrosive and will irritate skin on contact. Wear gloves and eye protection, and work in a chemical fume hood. |
Tris Base | Research Products International | T60040 | |
Triton X-100 | Sigma-Aldrich | T9284 | |
Vacuum centrifuge and vapor trap | Thermo Scientific | model nos. SpeedVac SPD120 and RVT5105 | |
Vortexer (Vortex-Genie 2) | Scientific Industries | ||
Water LC-MS | Honeywell | LC365-4 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved