A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

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 mitosis^1,2,3. 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 sites^2,3. 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 be performed for each cell line or tissue being used in an experiment¹⁸. If the sample is limited and 1 mg cannot be obtained,....

Results

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 treat.......

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 states^10,11,12,13. 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.......

Materials

List of materials used in this article

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