Oct 20, 2023

Public workspaceLiquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid Mass Spectrometer

DOI
dx.doi.org/10.17504/protocols.io.36wgq3m15lk5/v1
  • 1Buck Institute for Research on Aging
M A Watson
Open access
DOI: dx.doi.org/10.17504/protocols.io.36wgq3m15lk5/v1
Protocol Citation: J P Rose, M A Watson, B Schilling, Joanna Bons 2023. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid Mass Spectrometer. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3m15lk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 20, 2023
Last Modified: October 20, 2023
Protocol Integer ID: 89690
Keywords: LC-MS/MS, DIA, Orbitrap Eclipse, Proteomics, Mass Spectrometry
Abstract
Proteolytic peptide measurement using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid Mass Spectrometer for peptide/protein identification and quantification.
Materials

  • Dionex UltiMate 3000 liquid chromatographic system (Thermo Fisher Scientific)

  • Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific)

  • Acclaim PepMap 100 C18 trap column (75 µm x 2 cm, 3 µm particle size) (Thermo Fisher Scientific, PN 164535)

  • Acclaim PepMap 100 C18 analytical column (75 µm x 50 cm, 3 µm particle size; Thermo Fisher Scientific, PN 164570)

  • Autosampler vials and caps

  • LC-MS-grade water

  • Indexed retention time peptides (iRT, Biognosys)
  • Solvent A: 2% acetonitrile (ACN), 0.1% formic acid (FA) in water

  • Solvent B: 98% acetonitrile (ACN), 0.1% formic acid (FA) in water
Dilute the peptide sample with solvent A (2% ACN, 0.1% FA) to the desired concentration. Transfer the samples to an autosampler vial and add iRT peptides to the sample according to the manufacturer’s instructions.
Inject the sample onto a Dionex UltiMate 3000 liquid chromatographic system coupled to an Orbitrap Eclipse Tribrid mass spectrometer and load the peptides on a C18 trap column over 10 min at 5 µL/min with 100% solvent A.
Elute the peptides with a C18 analytical column heated at 35°C and at a flow rate of 300 nL/min over 215 min using the following gradient of solvent B:
2% for 10 min
From 2% to 20% in 125 min
From 20% to 32% in 40 min
From 32% to 80% in 1 min
80% for 9 min
From 80% to 2% in 1 min
2% for 29 min.
Acquire the data in data-independent acquisition (DIA) mode using the settings described in Table 1. One cycle consists of one full MS scan collected in the Orbitrap analyzer followed by DIA-MS/MS scans collected in the Orbitrap analyzer based on an isolation scheme of 26 variable windows covering the 350 - 1,650 m/z range with an overlap of 1 m/z (Table 2).

Table 1. Settings for data-independent acquisition on an Orbitrap Eclipse Tribrid mass spectrometer.
Table 2. Isolation scheme of the data-independent acquisition method.