Ultimate 3000 rslc nanolc

In solution, fractionation mass spectrometry (MS) was used to estimate the number of protein copies per cell based on the method described in Liu et al. (2019) (link). Cells were harvested and lysed in 600 µL solution containing 4% SDS, 100 mM Tris/HCl pH 7.6, and 0.1 M DTT. The lysate was incubated at 95°C for 5 min and then sonicated (20% power, 20 sec, 2 pulses). The lysate was clarified by centrifugation at 16,000g at room temperature for 5 min. Then 200 µL of cell lysate was prepared using the filter-aided sample preparation method. An Ultracel-10K (UFC501024; Millipore) filter was used for protein purification and trypsin digestion.
Mass spectrometry of the trypsinized peptides was performed by UT Southwestern proteomic core. An Ultimate 3000 RSLC nano-LC (Thermo Fisher Scientific) in-line connected to an Orbitrap Fusion Lumos (Thermo Fisher Scientific) was used for MS analysis. In brief, the sample was fractionated into 10 injections and peptides were loaded onto a reverse-phase column (Easy Spray column, either 75 µm × 50 cm or 75 µm × 75 cm, 2 µ beads). Raw files were processed using MaxQuant and used the latest human database from Uniprot. We then used the label-free quantization (LFQ, normalized intensity) data and histone signal to calculate the number of protein copies per cell.

The cleaned peptide mixtures were dried completely and resuspended in 20 μl of loading solvent [0.1% trifluoroacetic acid in water/acetonitrile, 2/98 (v/v)]. Two microliters of the peptide mixtures were analyzed by liquid chromatography (LC)–MS/MS on an UltiMate 3000 RSLCnano LC (Thermo Fisher Scientific, Bremen, Germany) in-line connected to a Q Exactive mass spectrometer (Thermo Fisher Scientific). Peptides were separated with a linear gradient at 300 nl/min from 98% solvent A (0.1% formic acid in water) to 55% solvent B [0.1% formic acid in water/acetonitrile, 20/80 (v/v)] in 120 min before ultimately reaching 99% solvent B. The mass spectrometer was operated in data-dependent, positive ionization mode, automatically switching between MS and MS/MS acquisition for the 10 most abundant peaks in a given MS spectrum.

30 μg of total protein of each sample (in triplicate) was prepared for LC-MS/MS analysis with the S-Trap® Micro Spin Column (ProtiFi, Farmingdale, NY, USA) digestion protocol according to the manufacturer’s instructions with slight modifications. 2 μg of purified peptides of each sample was then injected on an Ultimate 3000 RSLC nanoLC (Thermo Scientific®, Bremen, Germany) in-line connected to an LTQ-Orbitrap Elite (Thermo Fisher®). Detailed sample processing methodology and the mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [41] (link) partner repository with the dataset identifier PXD025633.