이전 사이트 이동

Carbamidomethyl (C)

Targeted proteomics

Thermo Scientific Q Exactive

Cells were lysed in 8 M urea in 50 mM Tris-HCl, pH 8.0 containing 1x HALT protease inhibitor cocktail (Thermo Scientific), and disrupted with BranSonic 400B sonifier. The lysate was cleared for 10 min at 10,000 g and 4 C. Proteins in the lysate were reduced (5 mM DTT, 45 min at 25C and 600 rpm), alkylated (20 mM 2-chloroacetamide, 45 min at 25 C and 600 rpm), and then diluted to bring the urea concentration to <0.8 M using 50 mM Tris-HCl, pH 8.0. Digestion was performed by adding trypsin (Promega, 1:50 enzyme-to-substrate ratio) and incubating overnight at 25 C and 600 rpm. Digests were acidified to pH < 3 by addition of trifluoroacetic acid (TFA) to 0.5% and were desalted using HLB solid-phase extraction (SPE) cartridges (Waters; wash solvent: 0.1% TFA; elution solvent: 0.1% FA in 50 % acetonitrile (ACN)). Eluates were dried by vacuum centrifugation and stored at -20 C.

PRM spectral assignment to each target used a two-step criterion: (i) MS1 precursor agreement within 0.001 m/z, and (ii) MS2 spectral matching to model-predicted fragments with a 5 ppm fragment-ion tolerance. PRM spectra exhibiting PCC values greater than 0.7, which represented the minimum PCC of PRTC standards, were selected, of which the spectra having at least 4 fragments were used for subsequent analysis. The intensities for fragment ions were integrated to get target peptide intensities and then normalized by the PRTC intensities to correct for run-to-run variation. The normalization was done using the crmn R package (v0.0.21).

A 2 µg peptide sample spiked with 125 fmol of PRTC peptide standard (Thermo Scientific) was injected onto a PepMap 100 trap column (75 µm x 20 mm, Thermo Fisher Scientific), washed with 0.1% FA in 2% ACN for 10 min at a flow rate of 5 µL/min and subsequently transferred to an EASY-Spray PepMap RSLC analytical column (75 µm x 150 mm, Thermo Fisher Scientific). Peptides were separated at 300 nL/min using a 55 min linear gradient from 2.0 to 24.0% LC solvent B (0.1% FA in 80% ACN) in LC solvent A (0.1% FA). Similar MS settings as described above were used, but the MS was operated in PRM mode with the following adjustments: PRM MS1 resolution was 70,000 at m/z 200 (Orbitrap), maxIT = 30 ms; targeted MS2 resolution = 35,000. Targeted MS2 spectra were recorded at a resolution of 35,000 and using an AGC target value of 2e5 charges, a maxIT of 200 ms, an isolation window of 1.2 m/z, and an isolation offset of 0.4 m/z. The number of targeted precursors per cycle was set to 20. The first mass was fixed to 200 m/z. Per six PRM LC-MS analyses, 0.5 µg of HeLa digest/PRTC standard (Thermo Fisher Scientific) was injected and analyzed with DDA method and identical LC gradients to PRM-MS. The inclusion list was comprised of 65 targets including 15 PRTC, 6 UPR related proteins, 21 Nt-arginylated peptides and 21 corresponding non-Nt-arginylated peptides. The charge state of each target peptide was determined using the “iep” application of EMBOSS. The PRM acquisition times for the targets were specified as -2 minutes for the start time and +4 minutes for the end time, based on the predicted RT. Out of 15 PRTCs, 14 except the most hydrophobic ones were used as internal standards (ISTD).