Label-free Quantitative Proteomics Workflow

A label-free proteomics approach was carried out in this research, which allows accurate relative quantification only between samples rather than within samples. Protein samples were precipitated with a solution of chloroform-methanol (2:1) and digested with trypsin. Afterwards, the samples were separated by C₁₈ reversed-phase liquid chromatography using a Waters NanoAcquity system and analyzed by mass spectrometry (MS) using a Thermo Orbitrap Fusion Lumos system. MS data were searched against the corresponding fungal databases from the Joint Genome Institute (JGI), and gene function was determined by Gene Ontology (GO) enrichment analysis with GOATOOLS (44 (link)) and manual curation. Protein quantification results were obtained with the MaxQuant software (version 1.6.1.0) with a mass error tolerance of 4.5 ppm. On the other hand, peptide quantitation and oxidative modifications were analyzed as dynamic modifications (45 (link)) with PEAKS Studio (version 10.0, build 20190129). A list of all the included PTMs is given in Table S1 in the supplemental material. Additional details are provided in Text S1.

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Castaño J., Zhang J., Zhou M., Tsai C.F., Lee J.Y., Nicora C, & Schilling J. (2021). A Fungal Secretome Adapted for Stress Enabled a Radical Wood Decay Mechanism. mBio, 12(4), e02040-21.

Publication 2021

Orbitrap Fusion Lumos system

Chloroform Gene function Genome Joint Mass spectrometry Methanol Peptide Protein Ptms Reversed phase liquid chromatography Tolerance Trypsin

Corresponding Organization : University of Minnesota

Other organizations : José Benito Vives de Andréis Marine and Coastal Research Institute, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory

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Variable analysis

independent variables

Protein samples were precipitated with a solution of chloroform-methanol (2:1)
Protein samples were digested with trypsin
Samples were separated by C18 reversed-phase liquid chromatography using a Waters NanoAcquity system
Samples were analyzed by mass spectrometry (MS) using a Thermo Orbitrap Fusion Lumos system
MS data were searched against the corresponding fungal databases from the Joint Genome Institute (JGI)

dependent variables

Protein quantification results
Peptide quantitation and oxidative modifications

control variables

Mass error tolerance of 4.5 ppm

controls

No positive or negative controls were explicitly mentioned.

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