Quantitative Proteomics by Data-Independent Acquisition

Data-independent acquisition quantitative proteomics was conducted according to a previous study (Zhang et al., 2021 (link)). Briefly, cells underwent protein extraction and trypsin digestion into peptides, and then a spectral library was generated and quantified. Ten fractions were collected, and each fraction was dried in a vacuum concentrator. The fractions were redissolved in 0.1% formic acid and analyzed using nanospray liquid chromatography with tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Lumos Tribrid (Thermo Fisher Scientific, MA, United States) coupled to a Waters nanoACQUITY UPLC System (Waters, MA, United States). The mass spectrometer was run in DDA mode and automatically switched between MS and MS/MS modes. The DDA data were processed and analyzed using Spectronaut X (Biognosys, Schlieren, Switzerland) with default settings to generate an initial target list. A false discovery rate cutoff at the precursor and protein levels was applied at 1%. Finally, proteins were identified and quantified.

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Feng L., Wang J., Zhang J., Diao J., He L., Fu C., Liao H., Xu X., Gao Y, & Zhou C. (2021). Comprehensive Analysis of E3 Ubiquitin Ligases Reveals Ring Finger Protein 223 as a Novel Oncogene Activated by KLF4 in Pancreatic Cancer. Frontiers in Cell and Developmental Biology, 9, 738709.

Publication 2021

Orbitrap Fusion Lumos Tribrid Waters nanoACQUITY UPLC System Spectronaut X

Cells Digestion Formic acid Library Liquid chromatography Ms ms Peptides Proteins Trypsin Vacuum

Corresponding Organization : Southern Medical University

Other organizations : The First Affiliated Hospital, Sun Yat-sen University, Sun Yat-sen University, Guangdong Provincial Hospital of Traditional Chinese Medicine, Gaozhou People's Hospital

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein identification and quantification

control variables

Protein extraction and trypsin digestion
Spectral library generation
Fractionation and drying of samples
Reconstitution in 0.1% formic acid
Nanospray liquid chromatography with tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Lumos Tribrid
Data-dependent acquisition (DDA) mode on the mass spectrometer
Data processing and analysis using Spectronaut X with default settings
False discovery rate cutoff at 1% for precursor and protein levels

controls

No positive or negative controls were explicitly mentioned in the provided information.

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