Membrane mass spectrometry

We employ mass spectrometry to study all components of biological membranes: Membrane and membrane-associated proteins, cofactors of membrane proteins and small molecules and lipids. We perform qualitative and quantitative proteomics analyses to characterize membrane proteins, identify and characterize ligands, perform functional studies on membrane proteins, and are currently establishing lipidomics analyses. For structural biologists, we have set up a MALDI-MS-based assay that allows the analysis of individual membrane protein crystals.

Membrane protein characterization
Using Peptide Mass Fingerprinting (PMF), we employ proteolytic digests of the target proteins to identify them by matching the acquired tandem-mass spectra to a proteome database. We have optimized our protocols for the analysis of membrane proteins, allowing us to obtain sequence coverage also of hydrophobic domains in membrane proteins.
In this context we have analysed cbb3 cytochrome c oxidase from Pseudomonas stutzeri Zobell, finding two previously uncharacterized isotypes for this strain. Our analyses also showed that the purification protocols allow selective isolation of only one isotype, a prerequisite for the subsequent crystallization of the protein (Buschmann et al., 2010).
We also identified differentially regulated proteins in a suppressor mutant of a CO-sensitive deletion variant of Methanosarcina acetivorans (Kliefoth et al., 2011).
In addition, we are setting up label-free quantitation workflows to analyse complex protein samples. In collaboration with the lab of Felix Wieland (Heidelberg University Biochemistry Center), we are currently investigating the role of the isotypes of the COPI coating protein coatomer in vesicular, intracellular transport. In this context, we have recently identified previously uncharacterized Arf-isoforms that also support COPI vesicle formation (Popoff et al., 2011).
In a project on synaptic plasticity with Erin Schuman (MPIH) we are using a “BONCAT”-based assay, a labelling strategy for the specific detection and quantification of newly synthesized proteins in acute slices of rat hippocampal neurons.

: Figure 1: Analyzing the sodium/proton binding site in c-subunits of ATPases: Deconvoluted mass spectra (maXis) of c-subunits and labeling efficiencies determined for c11- and c15-rotors in different conditions.

Functional studies
We also perform top-down analyses and functional studies on membrane proteins. We are currently working in a joint project with T. Meier (SB) and J. Faraldo-Gomez (TMB) to analyse the proton or sodium binding pockets of ATPase c-subunits. Based on the structures provided by the Meier group, models for cation binding and translocation are calculated in the Gomez group. Our contribution is to challenge this data using a biochemical, direct-infusion ESI-MS-based, quantitative assay that probes the protonation state of the binding site of the c-subunit. Here we established a method that allows faster, more sensitive and more accurate measurements than published previously. We confirmed the predictions for ion selectivity of the c-rings of Ilyobacter tartaricus and Spirulina platensis and were able to analyse ion binding even in lipid environments (see figure 1, Krah et al., 2010 and Pogoryelov et al., 2010).

: Figure 2: Single-crystal MALDI mass spectrum of Mvh (Autoflex III Smartbeam).

Single-crystal mass spectrometry (scMS)
In structural biology, the quality of the protein crystals used for diffraction data acquisition comprises the key factor for the structural analysis. To assess protein identity and quality, and to characterize the compounds comprising individual crystals, we established a method to analyse individual membrane protein crystals. Single-crystal MS (scMS) directly probes the crystals that are washed, dissolved and embedded in matrix: Thus, the proteins comprising the crystals are identified, modifications and the degree of modification (i.e. quantification of percentage of labeling of a protein) can be evaluated, and potential truncations can be detected. This assay precludes the both time- and money-intensive diffraction data acquisition of crystals comprised of unsuitable proteins.
Typically, single membrane protein crystals with side lengths of >50µm are suitable for scMS. We are currently optimising our protocols to faciliate measurements on very fragile crystals.

Lipidomics analyses
We are currently testing MS-based assays to analyse lipids bound to selected membrane proteins. After extraction of the lipids from the purified target proteins, we are performing direct infusions of the extracts using a chip-based ESI-source (Advion NanoMate) for qualitative and quantitative analyses.

Selected publications

Mayer F, Leone V, Langer JD, Faraldo-Gomez JD, Muller V. A c subunit with four transmembrane helices and one ion (Na+) binding site in an archaeal ATP synthase: implications for c ring function and structure. J Biol Chem. 2012 Sep 24. [Epub ahead of print].

Wang T, Langer JD, Peng G, Michel H. Isolation, functional characterization and crystallization of Aq_1259, an outer membrane protein with porin features, from Aquifex aeolicus. Biochim Biophys Acta. 2012 Jul 24. S. 1570-9639 (12) 00153-7.

Pogoryelov D, Klyszejko AL, Krasnoselska GO, Heller EM, Leone V, Langer JD, Vonck J, Müller DJ, Faraldo-Gómez JD, Meier T. Engineering rotor ring stoichiometries in the ATP synthase. Proc Natl Acad Sci USA. 2012 Jun 19;109(25):E1599-608. Epub 2012 May 24. PMID: 22628564.

Burkhardt J, Vonck J, Langer JD, Salzer R, Averhoff B. An unusual N-terminal ααβαββα fold of PilQ from T. thermophilus mediates ring formation and is essential for piliation. J Biol Chem. 2012 Jan 17. [Epub ahead of print] PMID: 22253437

Popoff V, Langer JD, Reckmann I, Hellwig A, Kahn RA, Bruegger B, Wieland FT. Several Arf isoforms support COPI vesicle formation. J Biol Chem. 2011 Oct 14; 286(41):35634-42.

Kliefoth M, Langer JD, Matschiavelli N, Oelgeschläger E, Rother M. Genetic analysis of MA4079, an aldehyde dehydrogenase homolog, in Methanosarcina acetivorans. Arch Microbiol. 2012 Feb; 194(2):75-85. Epub 2011 Jul 7.

Pogoryelov D, Krah A, Langer JD, Yildiz O, Faraldo-Gómez JD, Meier T. Microscopic rotary mechanism of ion translocation in the F(o) complex of ATP synthases. Nature Chem Biol. 2010 Dec;6(12):891-9. Epub 2010 Oct 24.

Marcia M, Langer JD, Parcej D, Vogel V, Peng G, Michel H. Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase. Biochim Biophys Acta. 2010 Nov;1798(11):2114-2123.

Buschmann S, Warkentin E, Xie H, Langer JD, Ermler U, Michel H. The structure of cbb3 cytochrome oxidase provides insights into proton pumping. Science. 2010 Jul 16;329(5989):327-30.

Krah A, Pogoryelov D, Langer JD, Bond PJ, Meier T, Faraldo-Gómez JD. Structural and energetic basis for H(+) versus Na(+) binding selectivity in ATP synthase F(o) rotors. Biochim Biophys Acta. 2010 Jun-Jul;1797(6-7):763-72.

Equipment
We are equiped with an analytical Dionex RSLC and a Proxeon easy-nanoHPLC coupled to a Bruker maXis ESI-q-TOF mass spectrometer, a Bruker Proteineer spotting robot and a Bruker Autoflex III Smartbeam MALDI-TOF/TOF mass spectrometer. For the lipidomics analyses, we employ an Advion NanoMate chip-based nanospray infusion system on the maXis. Since 12/2011, we also operate a Thermo Orbitrap Elite with Dionex RSLCnano UPLC systems.

Funding

BMBF: INSTRUCT start up grant

EU-INSTRUCT preparatory phase grant

Max-Planck Society

Deutsche Akademischer Austauschdienst

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