20220712 Top Down Deconvolution in Thermo FreeStyle

Thermo's "FreeStyle" software 1.8 , part of XCalibur 4.5, has been updated to support deconvolution of all proteomic tandem mass spectra in an LC-MS/MS experiment. This tutorial examines both DDA (data-dependent acquisition) and inclusion list experiments to reveal the way the instrument was configured from the RAW data. We then visualize and deconvolute the experiments via the Xtract algorithm.
The term "deconvolution" frequently covers many roles for top-down experiments at both MS and MS/MS levels, ranging among:
A. feature detection (recognizing persistent precursor chromatograms in MS signals)
B. scan averaging (when multiple MS/MS scans represent the same precursor)
C. charge state inference (based on the spacing of isotopes in MS scans)
D. charge reduction (modeling neutral or singly-charged masses, in some cases combining signals from different charge variants)
E. deisotoping (combining intensity across an isotopic envelope) PMID20855543.
Our two experiments are drawn from PXD032724:
H. acinonychis data-dependent acquisition: 20140314_F_EV_Hacin-E2-C4ZT_r120-r120-HCD20.raw
H. acinonychis inclusion-list acquisition: 20140725_F_EV_Hac2_repl3_Inclusion_HCD25.raw
1. Read the method description from the RAW files:
ThermoRawMetaDump.exe 20140314_F_EV_Hacin-E2-C4ZT_r120-r120-HCD20.raw
ThermoRawMetaDump.exe 20140725_F_EV_Hac2_repl3_Inclusion_HCD25.raw
2. Suggest alteration of Xtract Signal-to-Noise under Default Options to 1 rather than 3.
Suggest alteration of Xtract Charge Range of 1-50 rather than 5-50.
Suggest alteration of Xtract Min Num Detected Charge to 1 rather than 3.
3. Conduct Xtract All on inclusion list experiment:
20140725_F_EV_Hac2_repl3_Inclusion_HCD25.raw
4. Examine Xtracted inclusion list experiment:
20140725_F_EV_Hac2_repl3_Inclusion_HCD25_Xtract.Raw, precursor 634.8908: X2XXX2, Reports: Spectrum List
5. Conduct Xtract deconvolution on individual DDA scan:
20140314_F_EV_Hacin-E2-C4ZT_r120-r120-HCD20.raw, scan 924, precursor 1205.41: Q17X01
6. Comparing Xtracted scan in Xtract All of DDA experiment:
Jump to scan 924: That which was beautiful looks terrible! Look at precursor m/z: 1483.43; this is the wrong spectrum. The scan number has changed! Use the MSn Browser to find the MS/MS relating to precursor 1205.41 (now scan #917).