Sequential Fractionation Strategy Identifies Three Missing Proteins in the Mitochondrial Proteome of Commonly Used Cell Lines

Mitochondria
are undeniably the cell powerhouse, directly affecting
cell survival and fate. Growing evidence suggest that mitochondrial
protein repertoire affects metabolic activity and plays an important
role in determining cell
proliferation/differentiation or quiescence shift. Consequently, the
bioenergetic status of a cell is associated with the quality and abundance
of the mitochondrial populations and proteomes. Mitochondrial morphology
changes in the development of different cellular functions associated
with metabolic switches. It is therefore reasonable to speculate that
different cell lines do contain different mitochondrial-associated
proteins, and the investigation of these pools may well represent
a source for mining missing proteins (MPs). A very effective approach
to increase the number of IDs through mass spectrometry consists of
reducing the complexity of the biological samples by fractionation.
The present study aims at investigating the mitochondrial proteome
of five phenotypically different cell lines, possibly expressing some
of the MPs, through an enrichment–fractionation approach at
the organelle and protein level. We demonstrate a substantial increase
in the proteome coverage, which, in turn, increases the likelihood
of detecting low abundant proteins, often falling in the category
of MPs, and resulting, for the present study, in the identification
of METTL12, FAM163A, and RGS13. All MS data have been deposited to
the MassIVE data repository (https://massive.ucsd.edu) with the data set identifier MSV000082409
and PXD010446.