COmplexome Profiling ALignment (COPAL) reveals remodeling of mitochondrial protein complexes in Barth syndrome

Abstract

Motivation: Complexome profiling combines native gel electrophoresis with mass spectrometry to obtain the inventory, composition and abundance of multiprotein assemblies in an organelle. Applying complexome profiling to determine the effect of a mutation on protein complexes requires separating technical and biological variations from the variations caused by that mutation.

Results: We have developed the COmplexome Profiling ALignment (COPAL) tool that aligns multiple complexome profiles with each other. It includes the abundance profiles of all proteins on two gels, using a multi-dimensional implementation of the dynamic time warping algorithm to align the gels. Subsequent progressive alignment allows us to align multiple profiles with each other. We tested COPAL on complexome profiles from control mitochondria and from Barth syndrome (BTHS) mitochondria, which have a mutation in tafazzin gene that is involved in remodeling the inner mitochondrial membrane phospholipid cardiolipin. By comparing the variation between BTHS mitochondria and controls with the variation among either, we assessed the effects of BTHS on the abundance profiles of individual proteins. Combining those profiles with gene set enrichment analysis allows detecting significantly affected protein complexes. Most of the significantly affected protein complexes are located in the inner mitochondrial membrane (mitochondrial contact site and cristae organizing system, prohibitins), or are attached to it (the large ribosomal subunit).

Availability and implementation: COPAL is written in python and is available from http://github.com/cmbi/copal.

Supplementary information: Supplementary data are available at Bioinformatics online.

Fig. 1.

COPAL procedure for the alignment of two complexome profiling samples. (a) The distance, or ‘local cost’ between two gel slices is the sum of the absolute differences of the iBAQ values for all proteins on that slice. Here, i is an index for each protein, and n is the total number of proteins in the complexome profile, x and y are intensities of proteins from profile 1 and profile 2. (b) As is the case in classical time warping, the cost(xy) is the minimum of the local cost at point (x, y) plus one of three preceding costs: the one above it (x, y−1) corresponding to a slice (gap) inserted in the alignment of gel y, the one beside it (x−1, y), corresponding to a slice inserted in gel x, or the one diagonal from it (x−1, y−1) corresponding to no slice insertion. After building up the matrix in this manner the two gels are aligned by tracing back along the optimal warping path, as in standard dynamic time warping, thus obtaining the alignment with the minimal global cost

Fig. 2.

Clustering and alignment of nine complexome profiling examples. The colors refer to the three different BNE gels on which the samples were run. (a) single linkage clustering of the profiles based on the total costs obtained from the pairwise alignments. (b) Locations of inserted slices after multiple alignments of BTHS mitochondria and control complexome profiles. Complexomes from the same gels tend to have slices inserted at the same locations. (c) Images of complexome profiles of mitochondria from the same control on three different BNE gels, illustrating the overall shifts in migration profiles between gels, e.g. at slice 40. (d) Summed-up intensities (iBAQ values) of all mitochondrial proteins in the original, unaligned and non-normalized gels. The heatmaps were scaled to the maximum over all the gels. (e) Summed-up intensities of all mitochondrial proteins in normalized and aligned gels. The green boxes indicate the positions of the slices added in the alignment procedure

Fig. 3.

Migration pattern of oxidative phosphorylation proteins for controls (green) and TAZ mutation cell lines (red). (a) The summed intensities of all detected subunits of complexes I, III and IV show that the respiratory chain, including complex I that is part of the S₀–S₁ peak, remains intact in the BTHS mitochondria. Nevertheless, changes in the abundance of the complex III dimer, and S₂–S₄ supercomplexes are evident when comparing average profiles between BTHS mitochondria and controls. (b) Levels of the large isoform of complex I subunit NDUFV3 were severely reduced in all BTHS mitochondria profiles, with a small peak still visible at around ∼1500 kDa, which corresponds to the peak of supercomplex S₀–S₁. Note that there is a substantial amount of variation in the controls for NDUFV3 in the height of the high-mass shoulder diagnostic for supercomplexes S₂–S₄

Fig. 4.

TAZ mutations significantly affect the MICOS complex. (a) GSEA shows that all MICOS complex proteins, except MIC25 that has position 381, rank high on the list of mitochondrial proteins most affected by the TAZ mutations and are part of the so-called leading-edge subset in GSEA (Subramanian et al., 2005). (b and c) Aligned migration profiles of the MICOS proteins MIC60 and MIC19, respectively. The TAZ mutations lead to an increase in the expression of both proteins and to a small shift to a lower molecular mass of the complete complex from ∼700 to 600 kDa. For MIC19 they lead to a new peak around a molecular mass of 58 kDa, potentially reflecting a new subcomplex, possibly a MIC19 dimer as the predicted molecular mass of mature MIC19 is 23 kDa. (d) Average migration profile of all MICOS proteins and OPA1 in controls and in BTHS mitochondria. The BTHS mitochondria display, relative to controls, a higher expression of MICOS proteins and a shift to a lower mass of the MICOS complex. OPA1 changes from a broad distribution in the controls to a pronounced single peak in the BTHS mitochondria centered around 100 kDa

Fig. 5.

Increased presence of tRNA binding proteins in the 39S ribosomal subunit. The average of all detected proteins of the large 39S subunit of the mitochondrial ribosome and the average of the tRNA binding subcomplex of MRPL40, 46, 48 and 55. The tRNA binding subcomplex has shifted from being mainly a separate subcomplex in the controls to being part of the 39S subunit in the BTHS mitochondria. This provides an explanation for the higher abundance and mass of the 39S subunit in the BTHS mitochondria. Abundance profiles of the (sub)complexes have been rescaled to include them in the same panel. The combined molecular mass of these proteins together with the tRNA (∼120 kDa) closely matches that of the observed subcomplex in the figure. (b) Structure of the 39S subunit (PDB 3J9M) (Amunts et al., 2015) in which the tRNA binding proteins MRPL40, 46, 48 and 55 have been highlighted