Dec 30, 2025
Version 2
LC-MS/MS Proteomics Data Searching for PASEF DIA and DDA V.2
- hayes.mcdonald 1,
- Tina Tsui1,
- Jamie Allen1,
- Madeline E. Colley1,
- Melissa Farrow1,
- Jeff Spraggins1
- 1Vanderbilt University
Protocol Citation: hayes.mcdonald , Tina Tsui, Jamie Allen, Madeline E. Colley, Melissa Farrow, Jeff Spraggins 2025. LC-MS/MS Proteomics Data Searching for PASEF DIA and DDA. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk15pdg5r/v2Version created by Madeline E. Colley
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: December 30, 2025
Last Modified: December 30, 2025
Protocol Integer ID: 236048
Keywords: ms proteomics data, unlabeled proteomics data, search parameters for dia, mass spectrometer, dda pasef bottom, pasef dia, search parameter, dia
Disclaimer
Scaffold DDA and Spectronaut are paid software
Abstract
The following are search parameters for DIA and DDA PASEF bottom-up, unlabeled proteomics data collected on timsTOF mass spectrometers.
Before start
1. Scaffold DDA viewer can be downloaded for free here: www.proteomesoftware.com
DDA Searching with Scaffold sDDA version 6.7.4
A new project was made within Scaffold DDA version 6.7.4 with all samples loaded simultaneously
Data were searched within sDDA using MSFragger with the default parameters recommended by sDDA using the human uniprot database containing 50% decoys
With these initial filters, data were filtered within sDDA with 5% peptide and 5% protein FDR with a 2 peptide minimum
Data was then additionally filtered down to 1% peptide and 1% protein FDR with a 2 peptide minimum to ensure quality reports
Exports were generated from sDDA GUI view:
- Samples report
- Peptide report
- Spectra report by protein group
DIA Searching with Spectronaut version 20.3
Create a new project and load all DIA raw files into Spectronaut version 20.3
Search the data within spectronaut as a "library free" analysis bases on the human uniprot database with fully default parameters
Export the following pre-loaded report formats
- Pivoted BGS report
- Pivoted Protein report