Aug 15, 2023
Calculating mitochondrial protein solubility
- 1Lazarou Lab, WEHI
Protocol Citation: Louise Uoselis 2023. Calculating mitochondrial protein solubility. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxx7nlpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 15, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86481
Keywords: ASAPCRN
Abstract
Protocol for generating solubility calculations for mitochondrial proteins from starting 'soluble' and 'insoluble' fraction mass spectrometry data, using MaxQuant and Perseus software pipelines.
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 Tissue. Mol Cell Proteomics 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