Each individual sample was run in DIA mode using the same settings as the chromatogram library runs except using staggered isolation windows of 12 Da in the m/z range 400–1000 m/z. DIA data were analyzed using Scaffold DIA v.2.0.0 (Proteome Software, Portland, OR, USA). Raw data files were converted to mzML format using ProteoWizard v.3.0.11748 [39 (link)].
Scaffold dia v 2
Manufactured by Proteome Software
Sourced in United States
Scaffold DIA v.2.0.0 is a software tool designed for the analysis and visualization of data-independent acquisition (DIA) mass spectrometry data. It provides a platform for the processing, identification, and quantification of proteins from complex biological samples.
Automatically generated - may contain errors
5 protocols using scaffold dia v 2
1
DIA Mass Spectrometry Workflow
2
SWATH-based Proteomic Data Analysis Protocol
3
Peptide Identification and Quantification by DIA-MS
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Peptides were desalted and trapped on a Thermo PepMap trap and separated on an Easy-spray 100 μm × 25 cm C18 column using a Dionex Ultimate 3000 nUPLC at 200 nL/min. Solvent A = 0.1% formic acid, Solvent B = 100% Acetonitrile 0.1% formic acid. Gradient conditions = 2% B to 50% B over 60 min, followed by a 50–99% B in 6 min and then held for 3 min than 99% B to 2% B in 2 min and total run time of 90 min using Thermo Scientific Fusion Lumos mass spectrometer running in DIA mode. Six-gas phase fractionated (GPF) chromatogram library injections were made using staggered 4 Da isolation widows. GPF1 = 400–500 m/z, GPF2 = 500–600 m/z, GPF3 = 600–700 m/z, GPF4 = 700–800 m/z, GPF5 = 800–900 m/z, GPF6 = 900–1000 m/z, mass spectra were acquired using a collision energy of 35, resolution of 30 K, maximum inject time of 54 ms and a AGC target of 50 K.
Each individual sample was run in DIA mode using the same settings as the chromatogram library runs except using staggered isolation windows of 12 Da in the m/z range 400–1000 m/z. DIA data was analyzed using Scaffold DIA v.2.0.0 (Proteome Software, Portland, OR, USA). Raw data files were converted to mzML format using ProteoWizard v.3.0.1174843 (link). Total ion chromatograms can be found in S10 for CSF and S11 for Serum.
Each individual sample was run in DIA mode using the same settings as the chromatogram library runs except using staggered isolation windows of 12 Da in the m/z range 400–1000 m/z. DIA data was analyzed using Scaffold DIA v.2.0.0 (Proteome Software, Portland, OR, USA). Raw data files were converted to mzML format using ProteoWizard v.3.0.1174843 (link). Total ion chromatograms can be found in S10 for CSF and S11 for Serum.
4
DIA Mass Spectrometry Proteomics Protocol
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Each individual sample was run in DIA mode using the same settings as the chromatogram library runs except using staggered isolation windows of 12 Da in the m/z range 400-1000 m/z. DIA data was analyzed using Scaffold DIA v.2.0.0 (Proteome Software, Portland, OR, USA). Raw data files were converted to mzML format using ProteoWizard v.3.0.11748 [35] (link).
5
Peptide Separation and DIA Analysis
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Peptides were desalted and trapped on a Thermo PepMap trap and separated on an Easy-spray 100 µm x 25 cm C18 column using a Dionex Ultimate 3000 nUPLC at 200 nL/min. Solvent A = 0.1% formic acid, Solvent B = 100% Acetonitrile 0.1% formic acid. Gradient conditions = 2% B to 50% B over 60 minutes, followed by a 50%-99% B in 6 minutes and then held for 3 minutes than 99% B to 2% B in 2 minutes and total run time of 90 minutes using Thermo Scienti c Fusion Lumos mass spectrometer running in DIA mode. Six-gas phase fractionated (GPF) chromatogram library injections were made using staggered 4 Da isolation widows. GPF1 = 400-500 m/z, GPF2 = 500-600 m/z, GPF3 = 600-700 m/z, GPF4 = 700-800 m/z, GPF5 = 800-900 m/z, GPF6 = 900-1000 m/z, mass spectra were acquired using a collision energy of 35, resolution of 30 K, maximum inject time of 54 ms and a AGC target of 50K.
Each individual sample was run in DIA mode using the same settings as the chromatogram library runs except using staggered isolation windows of 12 Da in the m/z range 400-1000 m/z. DIA data was analyzed using Scaffold DIA v.2.0.0 (Proteome Software, Portland, OR, USA). Raw data les were converted to mzML format using ProteoWizard v.3.0.11748. 39 Total ion chromatograms can be found in S10 for CSF and S11 for Serum.
Each individual sample was run in DIA mode using the same settings as the chromatogram library runs except using staggered isolation windows of 12 Da in the m/z range 400-1000 m/z. DIA data was analyzed using Scaffold DIA v.2.0.0 (Proteome Software, Portland, OR, USA). Raw data les were converted to mzML format using ProteoWizard v.3.0.11748. 39 Total ion chromatograms can be found in S10 for CSF and S11 for Serum.
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