LILBID-mass spectrometry applied to the mass analysis of RNA polymerase II and an F1Fo-ATP synthase
Introduction
We recently developed a laser desorption mass spectrometry method [1] called LILBID (laser induced liquid bead ion desorption), in which biomolecules are laser desorbed/ablated from liquid microdroplets of solution. The method proved to be of nearly arbitrary softness, allowing to detect both specific weakly bound complexes at low laser intensity and also their covalent subunits at elevated intensity level. One of its major advantages is the low analyte consumption amounting to only few microliters of solution at micromolar concentration per analysis, making it an ideal tool for analysing biomolecules of low availability. This has been demonstrated recently for both large membrane-embedded molecules [2], [3] and specific complexes of DNA and RNA with ligands [4]. Another important advantage is the tolerance of the method to various buffers in solution, which often play a crucial role in determining binding selectivity and strength as e.g., in the case of DNA/RNA–ligand complexes [5]. Here we demonstrate the power of LILBID to analyze two large protein complexes: the water-soluble RNA polymerase II (m/z = 443 kDa) and a large membrane-inserted molecular machinery, the ATP synthase (m/z = 542 kDa) from Bacillus sp. strain TA2.A1. In the latter case, not only the mass of the integral complex but also mass fingerprints of the individual subunits are of great interest and play an important role in the characterization and structural analysis of this enzyme as recently demonstrated [3]. Both enzymes described here play a leading role in life processes: polymerase II is the transcriptional machinery of eukaryotic cells, transcribing DNA into mRNA, while the ATP synthase converts ADP and phosphate into ATP, the universal energy currency of all cells.
Section snippets
Method
LILBID is a mass spectrometry method which allows an exact mass determination of single macromolecules dissolved in droplets of solution containing an adequate buffer, pH, ion strength, detergents etc. The details of the setup have been described elsewhere [1], [5]. Briefly, droplets of solution of analyte are ejected by a piezo driven droplet generator. After their transfer into high vacuum they are detected droplet by droplet (ϕ = 50 μm, V = 65 pl, 10 Hz) by laser desorption/ablation. The laser is
Materials
The present species of polymerase II is a deletion version of the holo-polymerase II in which the two subunits Rpb4 and Rpb7 were removed. The polymerase was expressed and purified in the Lab of P. Cramer (LMU Munich) according to the purification procedure described [8]. The LILBID measurements were done with 1.6 μM polymerase II in 5 mM ammonium acetate at pH 7.5 or with 1.44 μM polymerase II with 100 mM ammonia at pH 10.5.
The F1Fo-ATP synthase from Bacillus sp. strain TA2.A1 was heterologously
RNA polymerase II
To demonstrate the power of LILBID to mass analyze large macromolecules we first studied RNA polymerase II, which is the central machinery in the transcription of DNA. Transcription is the process in which the information stored in a DNA is activated by synthesis of complementary mRNA by polymerases. RNA polymerase II (Pol II) is an enzyme found in eukaryotic cells. It is a 514-kDa complex and comprises 12 subunits with more than 28,000 atoms. Pol II is the best studied type of RNA polymerase
Conclusion
With LILBID-mass spectrometry we studied large macromolecules, which consist of many non-covalently bonded subunits. As example we first studied the water-soluble RNA polymerase II Δ, an enzyme involved in the transcription of DNA to mRNA and consisting of 10 subunits. The second example is an archetypical membrane-embedded protein complex. The ATP synthase, or complex V, we used in this study comprises in total 25 molecules forming a ATP producing nanomotor driven by the proton motive force
Acknowledgements
We gratefully acknowledge the lease of sample of polymerase II by Prof. J. Michaelis and Prof. P. Cramer (LMU Munich) and Prof. G. Cook (University of Otago, New Zealand) for critically reading the manuscript. TM was supported by the Cluster of Excellence “Macromolecular Complexes” at the Goethe University Frankfurt (DFG Project EXC 115).
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Cited by (17)
Biochemical Characterization of Cell-free Synthesized Human β<inf>1</inf> Adrenergic Receptor Cotranslationally Inserted into Nanodiscs
2022, Journal of Molecular BiologyCitation Excerpt :LILBID-MS depicts a native MS method allowing analysis of large protein complexes. During the process, increasing laser power partially disrupts the analyzed complexes and reveals all subunits and components (e.g. free GPCR, NDs without GPCR or monomeric MSP).14,15 The final data therefore indicate the largest complex in the sample, while simultaneously also detecting disintegration products.
Structure of the Human TRPML2 Ion Channel Extracytosolic/Lumenal Domain
2019, StructureCitation Excerpt :For native MS, the sample is transferred into a vacuum and desolvated by a mid-IR laser (Morgner et al., 2007). This has been successfully used to analyze the oligomeric state of large noncovalent protein complexes based on their disintegration pattern (Morgner et al., 2008; Peetz et al., 2018). In the MS spectra of the TRPML2 ELD, tetramer, dimer, and monomer, and, to a lesser extent, trimer species are observed (Figure 7D).
Cell-free expression and assembly of ATP synthase
2011, Journal of Molecular BiologyCitation Excerpt :The enzyme is encoded by a 7-kb atp operon, consisting of nine genes. The subunit stoichiometry of TA2-F1Fo is α3β3γδɛab2c13, with 9 soluble and 16 membrane-embedded proteins, which amount to a total mass of 542 kDa.27 The ATP synthase assembles completely, with all subunits correctly organized into the F1(α3β3γδɛ)Fo(ab2c13) complex.
Mass spectrometry of large complexes
2009, Current Opinion in Structural BiologyThree-dimensional structure of A<inf>1</inf>A<inf>0</inf> ATP synthase from the hyperthermophilic archaeon Pyrococcus furiosus by electron microscopy
2009, Journal of Biological ChemistryCitation Excerpt :Doubly charged states are visible for the c-subunit (the most abundant subunit) and the two catalytic proteins A and B, which are also highly charged in solution. A similar observation was made for these subunits in the F1F0 ATPase (50). The spectra contained some additional peaks that could not be assigned to any of the subunits.
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