Subunit mass fingerprinting of mitochondrial complex I

Abstract

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5-10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

Post-translational Modifications of Integral Membrane Proteins Resolved by Top-down Fourier Transform Mass Spectrometry with Collisionally Activated Dissociation

“…The "blue native" gel is probably the gold standard for separation of integral membrane protein complexes and might be the best option if ways can be devised to release complexes from the gel. This was achieved using electroelution with subsequent MS of the intact mitochondrial complex 1 using the laser-induced liquid bead ion desorption ionization technique, although it was noted that the St1 subunit was displaced from the complex apparently during running of the gel (27).…”

Truncation of subunit ND2 disrupts the threefold symmetry of the antiporter‐like subunits in complex I from higher metazoans

“…Finally, phylogenetic analyses of the subunit composition of eukaryotic complex I suggested that a number of supernumerary subunits are specific to higher metazoans [31]: if the presence of any of these subunits correlates with the truncation of ND2 then they may substitute structurally, if not functionally, for the deleted helices. Comparison of the well-defined subunit compositions of B. taurus and Y. lipolytica [7,32,33] identified nine B. taurus subunits that have no homologues in Y. lipolytica; four of them (subunits KFYI, MLRQ, SGDH, and B14.5b) are predicted to contain TMHs [7]. Thus, the sequences of these four subunits were compared, using blast searches, to extant sequence data from all the eukaryotes shown in Fig.…”

Three-dimensional Structure of A1A0 ATP Synthase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Electron Microscopy

“…Soft ion desorption conditions at low laser intensity allow the complex to be transferred intact into vacuum without fragmentation. The mass spectra were calibrated against the known subunits of complex I of Y. lipolytica, which are in the same mass range (51). The mass spectrum in Fig.…”