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TopDIA: A Software Tool for Top-Down Data-Independent Acquisition Proteomics

ABSTRACT: E. coli proteins (300 ng) extracted from the sample were analyzed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) coupled with an Ultimate 3000 (Thermo Fisher Scientific) reversed-phase liquid chromatography (RPLC) separation system with a C2 column (60 cm length, CoAnn Inc.). In the RPLC system, phase A was water with 0.1% formic acid (FA), and phase B was 60% acetonitrile (ACN) and 15% isopropanol (IPA) with 0.1% FA. A 98-min gradient of mobile phase B (0-5 min 5%, 5-7 min for 5% to 35%, 7-10 min for 35% to 50%, 10-97 min for 50% to 80%, 97-98 min from 80% to 99%) was applied with a flow rate of 400 nL/min. E. coli proteins were analyzed using both DDA and DIA modes. In each mode, six runs were performed separately, with each targeting a specific m/z range within the MS1 scan: 720-800, 800-880, 880-960, 960-1040, 1040-1120, and 1120-1200 m/z. MS1 spectra were collected with a resolution of 240,000 (at 200 m/z), 4 microscans, an automatic gain control (AGC) target value of 1x106, and a maximum injection time of 200 ms. MS/MS spectra were obtained with a scan range of 400-2000 m/z, a resolution of 60,000 (at 200 m/z), 1 microscan, an AGC target value of 1x106, and a maximum injection time of 500 ms. Fragmentation was performed using higher-energy collisional dissociation (HCD) with 30% NCE. In the DDA runs, the top six precursor ions from each MS1 scan were isolated with a 3 m/z window for MS/MS analysis. The dynamic exclusion was set to 60 seconds. In the DIA runs, a 4 m/z isolation window was used, resulting in a total of 20 MS/MS spectra for each cycle. Three technical replicates were obtained for each experiment.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Escherichia Coli K-12 (ncbitaxon:83333)

SUBMITTER: Xiaowen Liu

PROVIDER: MSV000094407 | MassIVE | Tue Mar 26 10:49:00 GMT 2024

REPOSITORIES: MassIVE

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TopDIA: A Software Tool for Top-Down Data-Independent Acquisition Proteomics

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