Proteome analysis

Steps

Mass spectrometry data was

analyzed according to a published protocol

Citation

Wingo TS, Duong DM, Zhou M, Dammer EB, Wu H, Cutler DJ, Lah JJ, Levey AI, Seyfried NT 2017 Integrating Next-Generation Genomic Sequencing and Mass Spectrometry To Estimate Allele-Specific Protein Abundance in Human Brain. https://doi.org/10.1021/acs.jproteome.7b00324

Spectra were searched using Proteome Discoverer (RRID:SCR_014477) (https://www.thermofisher.com/order/catalog/product/IQLAAEGABSFAKJMAUH) software version 2.1 against 2020 mouse UniProtKB/Swiss-Prot

(RRID:SCR_021164)( https://www.expasy.org/resources/uniprotkb-swiss-prot) (17,042 target sequences) along with the human α-synuclein protein sequence.

Searching parameters included full tryptic or Asp-N restriction, precursor mass tolerance

(± 20 ppm), and fragment mass tolerance (± 0.05 Da). Serine, threonine, and tyrosine

phosphorylation (+79.9663 Da), methionine oxidation (+15.99492 Da),

asparagine and glutamine deamidation (+0.98402 Da), and protein N-terminal

acetylation (+42.03670 Da) were variable modifications (up to 3 allowed per

peptide); cysteine was assigned a fixed carbamidomethyl modification (+57.021465

Da).

Percolator was used to filter the peptide spectrum matches to a false

discovery rate of 1%.

Gene ontology was analyzed using The Database for Annotation, Visualization and

Integrated Discovery DAVID (RRID:SCR_001881)https://david.ncifcrf.gov/tools.jsp-DAVID⁹⁷7.

Biological processes involving synaptic function were selected for grouping analysis. Functional grouping was based on Fisher’s exact test (p<0.05).

Protein-protein interaction networks were then identified using the STRING (RRID:SCR_005223 ) https://string-db.org//) version 11.5 , and the protein class was determined using PANTHER

(RRID:SCR_004869)( http://www.pantherdb.org/).

Abstract

Steps

推荐阅读