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

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.

PIB-MS is a novel technique used to enrich for endogenous phosphorprotein phosphatases and they're interacting proteins from cells and tissues. It involves the identification and quantification of these proteins using mass spectrometry-based proteomics. This technique does not require the exogenous expression of tagged versions of phosphorprotein phosphatases that could alter protein activity or localization.

Also, it does not use antibodies that might be non-specific or unable to distinguish between specific isoforms. This method for investigating the PPPM can be scaled for use on everything from cells to clinical samples. To begin, collect cell pellet by centrifugation at 277 times g for two minutes at room temperature.

Remove media and wash the cells with five milliliters of PBS. Prepare lysis buffer as described in the text protocol and keep on ice. Add one milliliter of chilled lysis buffer to the samples, then homogenize the samples by sonification and keep the cells on ice between pulses.

Transfer the lysates to fresh 1.5-milliliter tubes. Centrifuge the sonicated sample at 21, 130 times g for 15 minutes at 4 degrees Celsius. Use an aliquot of the lysate to quantify the total protein concentration using a bicinchoninic acid assay.

This step is essential to ensure equal protein concentration in each sample. For a phosphoprotein phosphatase inhibitor control, prepare two aliquots of equal protein concentration for each sample. Lysis buffer can be used to dilute the samples so that each tube has equal protein concentration.

Add one micromolar of free microcystin-LR to one aliquot and an equal volume of DMSO to the other. Vortex the samples and incubate them on ice for 15 minutes. Preparation of the phosphatase inhibitor beads should be carried out ahead of time or during the incubation steps described in the sample preparation section.

Use 10 microliters of solid phosphatase inhibitor beads, or PIB resin, for one milligram of protein and add them to a 1.5-milliliter tube. Wash the PIBs thrice with 0.5 milliliters of lysis buffer by vortexing followed by centrifugation at 376 times g for 30 seconds at room temperature. Add an appropriate amount of lysis buffer to the washed PIBs to make 50%by volume PIB buffer solution.

Mix by gently pipetting and swirl the pipette tip and the slurry to resuspend the PIBs. Transfer 20 microliters of the slurry to a 1.5-milliliter tube containing 0.5 milliliters of lysis buffer. Spin the tubes at 376 times g for 30 seconds.

Each tube contains equal volumes of PIBs in it. Discard the supernatant while leaving only the solid resin and up to 50 microliters of lysis buffer in each tube. Transfer the lysates to the appropriately labeled tubes containing PIBs.

Incubate the lysate with the PIBs for one hour at 4 degrees Celsius with rotation at 8 rpm. After incubation with the lysate, centrifuge the PIBs at 376 times g for 30 seconds at 4 degrees Celsius and remove the supernatant. Wash the beads by adding 0.5 milliliters of lysis buffer and inverting the tubes Collect the beads by centrifugation and remove the supernatant.

Repeat this step for a total of three washes. After the final wash, remove the lysis buffer as much as possible without pipetting up the PIBs. Prepare elution buffer containing 2%SDS and add the elution buffer four to five times the volume of PIBs.

Incubate at 65 degrees Celsius for one hour to elute the phosphoprotein phosphatases from the beads. Centrifuge the tubes at 376 times g for 30 seconds at room temperature. Collect the eluate into a separate tube and proceed for mass spectrometry analyses or western blotting.

To regenerate the PIBs, incubate them in 2%SDS solution with rotation at 8 rpm for one hour at room temperature. Wash the beads three to five times in 25 millimolar Tris-HCl pH 7.5 with rotation for 30 minutes per wash. Store PIBs in 25 millimolar Tris HCL pH 7.5 with sodium azide.

For mass spectrometry analysis, methods of data filtering and analysis vary and may be performed by the researcher or a core facility. After searching the raw data against a species-specific proteome database, filter the search results and export it as a Microsoft Excel worksheet. Analyze the data in triplicates.

For label-free quantification, use MS1 peak area measurements to quantify the data. To identify PPP subunits and their interactors de novo, compare biological triplicates of microcystin-LR-inhibited and DMSO-treated samples. Filter the data so that only proteins with a total peptide count of more than one in at least two of the three DMSO control-treated samples are present.

To filter out proteins that non-specifically bind to the resin, remove the proteins in which the total peptide count in the microcystin-LR-treated condition is higher than that for any PPP catalytic subunit. Exclude common contaminants such as keratin, collagen, ribosomal proteins and heterogeneous nuclear ribonucleoproteins from the analysis. A CSV file is essential for importing the data into Perseus.

Import filtered data into Perseus by clicking Generic matrix upload in the Load section. Transform the data by going to Basic Transform, selecting the data and specifying the transformation function as log base 2 of x. Impute missing values from a normal distribution by going to Imputation, Replace missing values from normal distribution, selecting the data and specifying the width and the downshift for the calculation.

Perform quantile normalization by going to Normalize, Quantile normalization. Annotate the data by going to Annot rows, Categorical annotation rows. Perform the student's T test by going to Tests, Two-sample tests, selecting the groups, the test to perform and the method for multiple hypothesis testing correction as used for truncation.

This function also calculates the log 2 ratios. Phosphoprotein phosphotates and their interactors were identified in the HeLa cells using a PIB pull-down experiment, followed by label-free quantification of protein abundance in DMSO-treated and microcystin-LR-treated samples. Volcano plot of mass spectrometry analysis identified specific PIB binders shown in red, catalytic subunits in blue, and new candidate PPP subunits, or interacting proteins, in white.

Non-specific binders were shown in gray. Scatter plot of log2 area from the DMSO-treated HeLa cell lysate showed that the abundances were highly correlated, indicating reproducibility of the data. Network analysis of all phosphorprotein phosphatase subunits and interacting proteins showed that these proteins were specific interactors in the PIB pull-down.

Global proteomics analyses could be performed and the results compared with the PIB analysis to determine if the PPPome composition is driven by PPP abundance or regulated by post translational mechanisms. PIB-MS is a broadly applicable tool that has allowed us to identify differences in PPP expression patterns under various conditions such as interphase versus mitotic samples or differences between cancer cell lines.