Genetic control of the HDL proteome

Nathalie Pamir; Calvin Pan; Deanna L. Plubell; Patrick M. Hutchins; Chongren Tang; Jake Wimberger; Angela Irwin; Thomas Q. de Aguiar Vallim; Jay W. Heinecke; Aldons J. Lusis

flow rate and 1.4 kV setting. HDL digests were introduced into the gas phase by ESI, positive ion mass spectra were acquired with a orbitrap mass spectrometer (Fusion, Thermo Electron Corp.) using datadependent acquisition (one MS survey scan followed by MS/MS scans of the 8 most abundant ions in the survey scan) with a 350-1400 m/z scan. An exclusion window of 30 s was used after 2 acquisitions of the same precursor ion (Pamir et al., 2016; Vaisar et al., 2007) . Protein identification. MS/MS spectra were matched against performed using the Comet search engine (version 2015.01 rev. 1) against a mouse UniProt database appended with yeast caboxypeptidase Y protein sequence (52,639 total sequences). The following Comet search parameters were applied: peptide mass tolerance of +/-20.0 ppm allowing for C13 isotope offsets, full tryptic digest allowing up to 2 missed cleavages, oxidized methionine variable modification, and carbamidomethyl cysteine static modification. The search results were subsequently processed though the Trans-Proteomic Pipeline (version 4.8.0) using the PeptideProphet and ProteinProphet tools using an adjusted probability of ≥0.90 for peptides and ≥0.95 for proteins. Each charge state of a peptide was considered a unique identification (Nesvizhskii et al., 2003) . We used the gene and protein names in the Entrez databases (National Center for Biotechnology Information; based on the nomenclature guidelines of the Human Gene Nomenclature Committee (http://www.gene.ucl.ca.uk/nomenlature) for human (Wain et al., 2002) and Mouse Genome Informatics (http://www.infromatics.jax.org.nomen/) guidelines (Davisson, 1994) to identify HDL proteins and to eliminate the redundant identifications of isoforms and protein fragments frequently found in databases used in proteomic analysis (Rappsilber and Mann, 2002) . This approach also permits cross-referencing of proteins from different species.