The mass spectrometry facility was established in October 2008 as a part of the Core Center G (ESFRI-INSTRUCT) and officially opened in February 2010.
We are equipped with a nano-LC (Proxeon easy-nLC) and an analytical UPLC (Dionex Ultimate 3000 RSLC) coupled to a high-resolution ESI-q-TOF mass spectrometer (Bruker maXis) and a MALDI TOF/TOF mass spectrometer (Bruker Autoflex III Smartbeam).
Bruker maXis with Proxeon easy-nLC (left) and Bruker Autoflex III Smartbeam (right).
Membrane mass spectrometry: Methods for analysing components of biological membranes
We have implemented and developed the following mass-spectrometry based methods in our lab:
1. "Top-down" characterization of proteins. Using either direct infusions or UPLC-purified samples, we perform qualitative and quantitative studies on both soluble and membrane proteins. We use this approach to identify and sequence unknown proteins (top-down sequencing), to investigate modifications and mutations of the target proteins (qualitative analysis) and to determine the relative amounts of modified and unmodified proteins in in vitro assays (quantitative studies).
2. "Bottom-up" identification and characterization of proteins: Using Peptide Mass Fingerprinting (PMF), we employ proteolytic digests of the target proteins to identify them by matching the aquired tandem-mass spectra to a proteome database. We have optimized the protocols for the identification of membrane proteins, and the cycles of data aquisition, processing and evaluation are performed fully automated. The direct connection between the nano-LC and the mass spectrometer via an ESI-sourge allows fast, online tandem-MS data aquisition. A typical data set for a tryptic digest of a Coomassie-stained SDS-PAGE band is recorded in 35 minutes, with a minimum sample amount of about 20fmols. We also employ this approach to analyse variants, isotypes and modifications of target proteins.
Example: We have analyzed the isotypes found in cbb3 cytochrome c oxidase crystals from Pseudomonas stutzeri (Link).
In addition, we are establishing a 2D-nanoLC-MS-based system. This "MudPit"- and "ITRAQ"-based assay allows both qualitative and quantitative studies of complex samples like whole-cell lysates containing more than 1000 proteins.
Example: In a collaboration with Felix Wieland (Heidelberg University Biochemistry Center) we investigate membrane protein cargo uptake into COPI-vesicles and the role of COPI-isotypes in the directionality of intra-Golgi transport.
3. Single-crystal protein analysis (scMS). As part of the Membrane Protein Core Center we perform mass spectrometric analyses of individual membrane protein crystals. Using this method we are able to identify the protein constituting the crystal, determine its subunit composition, and analyze it for truncations, mutations and modifications (see MALDI-scMS in figure). Tyically, 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.
Example: Single-crystal MALDI mass spectrum of Methylviologen reducing hydrogenase from Methanothermobacter marburgensis showing all three subunits of the complex and allowing identification of isotype D1 (figure 2, crystal by Kristian Parey and Dr. Ulrich Ermler).
4. Small molecules: Lipids, cofactors and detergents. In order to identify, analyze and quantify lipids, cofactors, modifications and crosslinkers on target proteins, we perform analyses of organic phase extracts of target proteins.
Example: Region of interest in an ESI-MS of an organic phase extract of a target protein from Aquifex aeolicus (Ye Gao). The spectrum aquired on the maXis was unambigously matched to Heme b (isotope intensity pattern, see figure 3).
In addition, we are planning to implement on the nano-scale a quantitative lipid analysis system that has been established in our laboratory.
Zhang C, Allegretti M, Vonck J, Langer JD, Marcia M, Peng G, Michel H. Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli. Biochim Biophys Acta. 2013 Sep 1. [Epub ahead of print] PMID: 24005236
Zhang C, Marcia M, Langer JD, Peng G, Michel H. Role of the N-terminal signal peptide in the membrane insertion of Aquifex aeolicus F1F0 ATP synthase c-subunit. FEBS J. 2013 Jul;280(14):3425-35.
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.
BMBF: INSTRUCT start up grant
EU-INSTRUCT preparatory phase grant
Dr. Julian Langer
Phone: +49 (0) 69 6303 1062
Fax: +49 (0) 69 6303 1002