Peptide Preparation and Proteomic Analysis

Samples were prepared using the Thermo EasyPep Mini MS Sample Prep Kit (Thermo Scientific, A4006) according to the manufacturer’s instructions. The dried peptides were then dissolved in 0.1% formic acid (Thermo Scientific, 5178) solution, and peptide concentration was tested using the Pierce Quantitative Fluorometric Peptide Assay (Thermo Scientific, 23290). LC-MS/MS experiments were performed on an EASY-nLC 1000 (Thermo Fisher Scientific, Waltham, MA, USA) connected to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). Proteomic analysis was performed using Proteome Discoverer 2.4 (Thermo Scientific) software, the Uniprot human database and SequestHT with Percolator validation. The detailed information for LC/MS processing and data analysis was described previously [59 (link)].

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Wang F., Li S., Cheng K.W., Rosencrans W.M, & Chou T.F. (2022). The p97 Inhibitor UPCDC-30245 Blocks Endo-Lysosomal Degradation. Pharmaceuticals, 15(2), 204.

Publication 2022

Pierce Quantitative Fluorometric Peptide Assay EASY-nLC 1000 Orbitrap Eclipse Tribrid mass spectrometer Proteome Discoverer 2.4

Assay Fluorometric Formic acid Human Ms ms Peptide Proteome

Corresponding Organization : California Institute of Technology

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Variable analysis

independent variables

Sample preparation method: Thermo EasyPep Mini MS Sample Prep Kit
Peptide dissolution: 0.1% formic acid solution
Peptide concentration measurement: Pierce Quantitative Fluorometric Peptide Assay

dependent variables

LC-MS/MS experimental results
Proteomic analysis using Proteome Discoverer 2.4 software

control variables

Uniprot human database
SequestHT with Percolator validation

controls

No positive or negative controls explicitly mentioned

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