Cell lysate preparation. Cells were harvested by counting each cell line in quadruplicate and were resuspended in a known volume of lysis buffer [8 M urea, 1% CHAPS, and 20 mM Hepes (pH 8.0)]. The suspension was pulsed six times with a sonic dismembrator on ice and was rotated end over end for 1 hour at 4°C. DNA was pelleted, and cell lysate was prepared for MS analysis.

Cell lysates were prepared using the filter-aided sample preparation (FASP) method. Briefly, 50 to 200 μg of protein per sample, at a concentration of 1 to 10 μg/μl, was reduced with DTT (final concentration of 3 mM) at 57°C for 1 hour and loaded onto a MicroCon 30-kDa centrifugal filter unit pre-equilibrated with 200 μl of FASP buffer A [8 M urea and 0.1 M tris-HCl (pH 7.8)] (Millipore). Following three washes with FASP buffer A, lysates were alkylated on a filter with 50 mM iodoacetamide for 45 min in the dark. Filter-bound lysates were then washed three times each with FASP buffer A and FASP buffer B [100 mM ammonium bicarbonate (pH 7.8)] and were digested overnight at room temperature with trypsin (Promega) at a 1:100 ratio of enzyme to protein. Peptides were eluted twice with 0.5 M NaCl. Tryptic peptides were desalted using an UltraMicro Spin Column, C18, (Harvard Apparatus), and the desalted peptide mixture was concentrated in a SpeedVac concentrator.

Targeted MS assay development. Peptides used in this assay were selected on the basis of a series of experiments. First, we compiled data from the Global Proteome Machine Database (GPMDB) for each of the three proteins (41). GPMDB is a repository of the results of MS-based proteomics experiments. The data collected included the peptides and the charge states identified, their uniqueness to the protein, and the number of times that each was identified. The most often identified unique peptides and charge states of EED, EZH2, and SUZ12 were compared to the results of the recombinant analysis to generate a list of approximately five candidate peptides per protein.

Next, an equal molar trypsin-digested mixture of the three recombinant mouse PRC2 proteins (EED, EZH2, and SUZ12) was analyzed on a Q Exactive mass spectrometer (Thermo Fisher Scientific) using a data-dependent method. A list of identified proteotypic peptides was compiled. This list of peptides was used to generate a target list in the Skyline Software, which was used to build the targeted MS2 method. An aliquot of an mESC lysate was analyzed using this method. Raw files were uploaded to the Skyline Software. Candidate peptides were evaluated on the basis of the chromatographic peak shape, ionization efficiency, coverage of fragmentation, level of interference from other peptides within the isolation window, and the presence of product ions that differentiate the endogenous and heavy-labeled peptide. Two to three of the strongest candidate peptides were custom synthesized (New England Peptide) with an isotopically heavy C-terminal residue.

Targeted MS analysis. The peptides were spiked into an aliquot of the cell lysate at a level ranging from 100 amol to 5 fmol. The peptide mixture was loaded onto an Acclaim PepMap trap column in line with an EASY-Spray PepMap C18 analytical high-performance liquid chromatography (HPLC) column (inner diameter, 50 cm by 75 μm) with a 2-μm bead size using the auto sampler of an EASY-nLC 1000 HPLC (Thermo Fisher Scientific). The peptides were gradient eluted into a Q Exactive mass spectrometer (Thermo Fisher Scientific) using a 60-min gradient from 5 to 23% of solvent B (solvent A, 2% acetonitrile and 0.5% acetic acid; solvent B, 90% acetonitrile and 0.5% acetic acid), followed by 20 min from 23 to 45% of solvent B. Solvent B was taken to 100% in 10 min and held at 100% for 20 min. High-resolution full MS spectra were acquired with a resolution of 70,000, an automatic gain control (AGC) target of 1 × 106, a maximum ion time of 120 ms, and a scan range of 300 to 1500 mass/charge ratio (m/z). Following each full MS scan, targeted high-resolution high-energy collisional dissociation tandem MS (MS/MS) spectra were acquired on the basis of the inclusion list containing the m/z values of the unlabeled endogenous and stable isotope–labeled standard PRC2 peptides. All MS/MS spectra were collected using the following instrument parameters: resolution of 17,500, AGC target of 2 × 105, maximum ion time of 120 ms, one microscan, isolation window of 2.0 m/z, fixed first mass of 150 m/z, and normalized collision energy of 27. Peptides not behaving linearly in lysate were excluded from future quantitation.

Quantitative experiments were conducted in two ways: (i) five-point calibration curve (500 amol to 5 fmol) of standard peptides spiked into a constant amount of lysate or (ii) a single-point calibration of standard peptides spiked into a constant amount of lysate in triplicate in a concentration close or equal to the endogenous level.

Data analysis and validation. Raw files were uploaded to Skyline Software (64 bit) version 3.6.0.10162 for analysis. The top three or four product ions were used to quantify the light (endogenous) and stable isotope–labeled standard peptides (see data file S1). Total product ion area values were calculated for the light and heavy peptides.

When a calibration curve was generated, the total product ion area values for each of the stable isotope–labeled standard peptides were plotted, a linear trendline was fit to the data, and the line equation was used to calculate the femtomoles of endogenous peptide in the lysate. The average, SD, and the coefficient of variance across the triplicates are reported for all lysates.

To calculate the number of each PRC2 subunit (EZH2, EED and SUZ12) per cell, the average femtomoles is for each peptide quantified of that subunit were computed and converted to number of molecules using Avogadro’s number and was divided by the number of cells analyzed to obtain molecules per cell. Total PRC2 per cell was calculated by taking the average of all subunits.

A few attempts have been made to infer the number of PRC2 molecules per nucleus using a variety of imaging and enzyme kinetic techniques. Reports vary from numbers as low as 6000 [acute lymphoblastic leukemia/myeloma; (42)] to ~40,000 [Drosophila neuroblast; (43)] and as high as 20,006,965 in a Drosophila salivary gland (44). While our methods provided slight variations on these numbers, they are well within the range and ordinal differences reported, adding validity to these various methods of PRC2 quantitation.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.