Easy nlc 1000 nanoflow lc

LC−MS/MS was used for quantitative analysis of TOMM34 expression. All samples were collected at West China Hospital with the approval of the research ethics committee. LC−MS/MS analysis was performed with a TMT-based isobaric labeling strategy. Sample processing included protein extraction and digestion, isobaric labeling of TMT 10plex reagents (Thermo Scientific), peptide fractionation by high-performance liquid chromatography (HPLC), and desalting and loading of the sample onto a trap column and an analytical column for LC−MS/MS analysis. LC−MS/MS analysis was performed by an EASY-nLC 1000 nanoflow LC instrument coupled to a Q Exactive Plus Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific).

The LC‐MS/MS analysis was carried out with a Q Exactive ESI‐quadrupole‐orbitrap mass spectrometer coupled to an EASY‐nLC 1000 nanoflow LC (Thermo Fisher Scientific), using the Xcalibur version 3.1.66.10 (Thermo Scientific). The tryptic peptide sample mixture was loaded from autosampler into a C18‐packed precolumn (Acclaim PepMap™100 75 μm × 2 cm, 3 μm, 100 Å, Thermo Scientific) in buffer A (1% acetonitrile, 0.1% formic acid). Peptides were transferred onward to C18‐packed analytical column (Acclaim PepMap™100 75 μm × 15 cm, 2 μm, 100 Å, Thermo Scientific) and separated with a 120‐min linear gradient from 5% to 35% of buffer B (98% acetonitrile, 0.1% formic acid) at a flow rate of 300 nl/min. MS analysis was performed in a data‐dependent acquisition in a positive‐ion mode. MS spectra were acquired from m/z 200 to m/z 2,000 with a resolution of 70,000 with a full AGC target value of 1,000,000 ions and a maximal injection time of 100 ms in the profile mode. The 10 most abundant ions for which the charge states were 2+ to 7+ were selected for subsequent fragmentation (higher energy collisional dissociation (HCD), and MS/MS spectra were acquired with a resolution of 17,500 with an AGC target value of 5,000, a maximal injection time of 100 ms, and the lowest mass fixed at m/z 120, in the centroid mode. The dynamic exclusion duration was 30 s.