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    Proteome analysis

    Leonardo A Parra-Rivas

    Published: 2023-11-12 DOI: 10.17504/protocols.io.5jyl8pw86g2w/v1
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    Abstract

    Proteome analysis

    Steps

    1.

    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. http://doi.org/10.1021/acs.jproteome.7b00324

    2.

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

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

    3.

    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).

    4.

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

    discovery rate of 1%.

    5.

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

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

    6.

    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 ) http://string-db.org//) version 11.5 , and the protein class was determined using PANTHER

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

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