Calculating mitochondrial protein solubility

Process raw instrument files using MaxQuant (protocol performed with v1.6.17) with the Andromeda search engine, searching against the Uniprot human data base containing reviewed and canonical isoform variants in the FASTA format, with recombinant Ag85A sequence added as a custom entry to the human database

Set the LC-MS run to "Reporter ion MS"

Set TMT11-plex labels as isobaric labels with a reporter ion mass tolerance of 0.003 Da

Set Trypsin/P cleavage specificity to a maximum of 2 missed cleavages

Set oxidation of methionine and N-terminal acetylation as variable modifications, and carbamidomethylation of cysteine as a fixed modification

Enable the 'Match between runs' option, with an FDR o f`% and mass bin size of 0.0065 Da

Set minimum unique and razor peptides to 1, and label min ratio count to 2

Run the MaxQuant analysis

Extract the proteinGroups.txt table, and normalise the data following the protocols outlined by:

D. L. Plubell, P. A. Wilmarth,

Y. Zhao, A. M. Fenton, J. Minnier, A. P. Reddy, J. Klimek, X. Yang, L. L.

David, N. Pamir, Extended Multiplexing of Tandem Mass Tags (TMT) Labeling

Reveals Age and High Fat Diet Specific Proteome Changes in Mouse Epididymal

Adipose Tiss Mol Cell Proteomics m 16 s 16, 873-890 (2017).

Average the internal spiked control (Ag85A) intensities across each reporter channel, and use this value to generate a scaling factor for each channel to normalise reporter ion intensities for each protein to the relative starting intensities in each sample at the time of Ag85A addition

Impute the data into Perseus and remove 'only identified by site', 'reverse', and 'potential contamination' identifications

Filter the clean data according to mitochondrial associated proteins, using a database such as the database found in:

I. Kuznetsova, A. Lugmayr, O. Rackham, A. Filipovska, OmicsVolcano: Software for intuitive visualization and interactive exploration of high-throughput biological data. STAR Protocols 2 , 100279 (2021).

Generate computationally derived 'total' fraction intensities by addition of the 'soluble' and 'insoluble' total fraction intensities

Calculate the percentage of insoluble protein for each protein by dividing the insoluble protein intensity by the calculated total protein intensity and multiplying the outputted value by 100