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 regulation of dynamic cellular processes is still emerging.
Phosphorylation dynamics are controlled by the dynamic interplay between kinases and phosphatases. In mammalian cells, there are more than 400 protein kinases that catalyze serine/threonine phosphorylation. Over 90% of these sites are dephosphorylated by phosphoprotein phosphatases (PPPs), a small family of enzymes that consists of PP1, PP2A, PP2B, PP4-7, PPT, and PPZ2,3. PP1 and PP2A are responsible for the majority of phosphoserine and phosphothreonine dephosphorylation within a cell2,3,4. The notable difference in number between kinases and phosphatases and the lack of specificity of PPP catalytic subunits in vitro led to the belief that kinases are the main determinant of phosphorylation2,3. However, multiple studies have shown phosphatases to establish substrate specificity through the formation of multimeric holoenzymes5,6,7,8,9. For example, PP1 is a heterodimer that consists of a catalytic subunit and, at a given time, one out of the more than 150 regulatory subunits6,7,8. Conversely, PP2A is a heterotrimer that is formed of a scaffolding (A), a regulatory (B), and a catalytic (C) subunit2,3,9. There are four distinct families of PP2A regulatory subunits (B55, B56, PR72, and striatin), each with multiple genes, splice variants, and localization patterns2,3,9. The multimeric nature of PPPs fills the gap in the number of kinases and PPP catalytic subunits. However, it creates analytical challenges for studying PPP signaling. To comprehensively analyze PPP signaling, it is critical to investigate the various holoenzymes within a cell or tissue. Great advances have been made in studying the human kinome through the use of kinase inhibitor beads, termed multiplex inhibitor beads or kinobeads, a chemical proteomic strategy where kinase inhibitors are immobilized on beads and mass spectrometry is used to identify enriched kinases and their interactors10,11,12,13.
We have established a similar approach to study PPP biology. This technique involves affinity capture of PPP catalytic subunits using beads with an immobilized, non-selective PPP inhibitor called microcystin-LR (MCLR) termed phosphatase inhibitor beads (PIBs)14,15. Unlike other methods that require the endogenous tagging or expression of exogenous PPP subunits that could alter protein activity or localization, PIB-MS allows for the enrichment of endogenous PPP catalytic subunits, their associated regulatory and scaffolding subunits, and interacting proteins (termed the PPPome) from cells and tissues at a given time point or under specific treatment conditions. MCLR inhibits PP1, PP2A, PP4-6, PPT, and PPZ at nanomolar concentrations, making PIBs highly effective at enriching for the PPPome16. This method can be scaled for use on any starting material from cells to clinical samples. Here, we describe in detail the use of PIBs and mass spectrometry (PIB-MS) to efficiently capture, identify, and quantify the endogenous PPPome and its modification states.
Figure 1: Visual summary of the PIB-MS protocol. In a PIB-MS experiment, samples can be obtained in various forms, from cells to tumors. The sample is collected, lysed, and homogenized prior to PPP enrichment. To enrich for PPPs, the lysate is incubated with PIBs with or without a PPP-inhibitor, such as MCLR. The PIBs are then washed, and PPPs are eluted in denaturing conditions. The samples are prepared for mass spectrometry analysis by the removal of detergents through SP3 protein enrichment, tryptic digestion, and desalting. Samples can then be optionally TMT-labeled prior to mass spectrometry analysis. Please click here to view a larger version of this figure.
PIB-MS involves lysis and clarification of cells or tissues, incubation of the lysate with PIBs, elution, and analysis of the eluate via western blotting or mass spectrometry-based approaches (Figure 1). The addition of free MCLR can be used as a control to distinguish specific PIB binders from non-specific interactors. For most applications, a label-free approach can be used to directly identify proteins in eluates. In cases where greater precision in quantification or the identification of low-abundance species is needed, further processing with tandem mass-tag (TMT) labeling can be used to increase coverage and decrease input.
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, the amount of input can be reduced with a minor loss of PPP subunit detection. Alternatively, TMT labeling can be employed to allow the mixing of all conditions in one sample, increasing the sensitivity of detection as shown in Step 9.
2. Preparation of PIBs
3. Incubation of PIBs with lysates
4. Washing of PIBs
5. Elution of PPPs from the PIBs
6. Removal of detergents
NOTE: Various approaches can be used to remove detergent from eluate samples for MS analysis. We found that single-pot, solid-phase-enhanced sample-preparation (SP3), described by Hughes et al., works well19.
7. Digestion of proteins
8. Desalting the digest
9. TMT labeling
NOTE: Tandem-mass tag labeling is used to multiplex samples for quantitative analysis. A 0.8 mg vial of TMT reagent is sufficient for labeling up to 0.8 mg of protein21. In a PIB pulldown experiment starting with 1 mg of protein, 1-3 µg of phosphoprotein subunits are obtained. The protocol below is optimal for up to 10 µg of protein.
10. Desalting the TMT-labeled combined sample
11. Data analysis
NOTE: Methods of data filtering and analysis vary and are beyond the scope of this protocol, but the following notes on analysis are included to provide guidance specific to the type of data resulting from this protocol.
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 analysis in DMSO and MCLR-treated HeLa cell lysates, with specific PIB binders shown in red. PPP catalytic subunits are labeled and shown in blue. New candidate PPP subunits or interacting proteins are shown in black. Non-specific binders are shown in grey. (C) Scatter plot of log2 area of two biological replicates from HeLa cell lysates treated with DMSO to demonstrate the reproducibility of the enrichment. Specific binders to PIBs are shown in red. Please click here to view a larger version of this figure.
Figure 3: Network analysis of all PPP subunits and PPP-interacting proteins identified in HeLa cells. These proteins were found to be specific PIB interactors in the HeLa PIB pulldown with and without MCLR. Please click here to view a larger version of this figure.
To demonstrate the performance of the PIBs, we performed a PIB pulldown experiment in HeLa cells to identify PPPs and their interactors (Figure 2A). HeLa cells were grown in biological triplicate and lysed. Each triplicate lysate was split in half, where half was treated with MCLR for 15 min to prevent the binding of PPP catalytic subunits or DMSO before PIB enrichment was performed. Following PIB enrichment, a label-free comparison of the abundance of proteins quantified in the DMSO-treated and MCLR-treated samples was performed to distinguish specific from non-specific interactors (Figure 2B). In the analysis, we detected all MCLR-sensitive PPP catalytic subunits PP1ca, PP1cb, PP1cc, PP2Aca, PP2Acb, PP4c, PP5c, and PP6c. In the DMSO-treated samples, the abundances (log2 areas of protein quantification) were highly correlated, indicating reproducibility (Figure 2C), yet upon treatment with MCLR, the PPP catalytic subunit or PPP interacting protein abundance in PIB pulldowns was greatly reduced (Figure 2B). In this analysis, we identified 92 PPP subunits and specific PPP interactors (see Supplementary Table 1), which is comparable to a previous PIB-MS analysis in HeLa cells14 (Figure 3).
Supplementary Table 1: MS data of representative PIB-MS analysis. Please click here to download this Table.
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 regulation of cellular dephosphorylation. Here, we show that, using PIBs, we can enrich catalytic and noncatalytic components of PP1, PP2A, PP4, PP5, and PP6. We are also able to identify new candidate PPP interactors in a variety of cell lines, tissue types, and organisms.
There are several critical steps in the PIB-MS protocol that should not be overlooked. These include the incorporation of appropriate controls and replicates, the input of equal amounts of protein across all samples, the maintenance of non-denaturing conditions during sample preparation, the incubation of the sample with PIBs, and the washing of the PIBs. With respect to controls and replicates, it is important that half of each sample is treated with a PPP inhibitor if the goal is to identify novel PPP subunits and interactors. This is required to differentiate between specific and non-specific PIB interactors. If seeking to identify how known PPP subunit or PPP interactor expression changes across cell or tissue types and cell cycle phases, or upon various drug treatments, it is critical to curate a list of known PPP subunits and interactors for comparison. Furthermore, these experiments are typically done in biological triplicate to ensure statistical confidence. Equal protein input per sample is essential for data reproducibility across samples; it also ensures that differences in PPP subunit or interactor abundance are not simply due to differences in protein input. A protein assay such as a BCA must be used to determine protein concentration in each sample. Lastly, non-denaturing conditions in sample preparation, PIB enrichment, and PIB washing is critical to ensure that the proteins remain in their native state. This preserves PPP subunit interactions.
PIB-MS is a powerful tool for interrogating the PPPome, yet it comes with several limitations. PIBs can enrich for the catalytic and noncatalytic components of PP1, PP2A, PP4, PP5, and PP6, but not PP2B or PP7, since MCLR does not inhibit these phosphatases at nanomolar concentrations. It is important to note that this tool is suited for ease of identification and quantification of endogenous, MCLR-sensitive PPP holoenzymes, such as those noted. PIB-MS is unable to detect PPP holoenzymes that are inhibited by endogenous PPP inhibitors that block the PPP active site, such as α4 and SET14. This technique does not require the exogenous expression of tagged PPP subunits or the use of PPP-specific antibodies. Like other affinity enrichment strategies using sepharose-based matrices, PIB-MS is prone to the binding of non-specific proteins. However, the addition of free MCLR as a negative control allows for distinguishing specific from non-specific interactors, enabling the identification of new PPP subunits and interacting proteins. Furthermore, non-specific interactors can be reduced via additional washes and the careful use of the appropriate amount of PIBs.
We have employed PIB-MS to compare the PPP expression patterns of various cancer cell lines, including breast cancer and glioblastoma14. We found that these cancer types have unique PPP expression patterns indicative of their origin14. PPPs are among the most conserved enzymes2,3. We were able to demonstrate that PIB-MS is effective in analyzing the PPPome in yeast and mouse tissues14. PIB-MS can also be used to identify differences in PPP expression patterns under various conditions, such as interphase versus mitotic samples15. Thus, PIB-MS provides insights into phosphoprotein phosphatase networks and expands our understanding of PPP regulation across multiple cell lines and disease states, as well as upon drug treatments.
The authors have nothing to disclose.
A.N.K. acknowledges support from NIH R33 CA225458 and R35 GM119455. We thank the Kettenbach and Gerber labs for their helpful discussion.
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 |