Customer InsightsFrom Individual Membrane Protein Complexes to in vivo Proteome Dynamics: Developing New Mass Spectrometry Solutions

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From Individual Membrane Protein Complexes to in vivo Proteome Dynamics: Developing New Mass Spectrometry SolutionsHigh speed MALDI mass spectrometry instrumentation is an essential element in multidisciplinary proteomics research at the Max Planck Institutes for Biophysics and Brain Research

A joint venture of the Max Planck Institutes of Biophysics and Brain Research

Julian Langer’s lab was established as a collabora-tive center between the Max Planck Institutes for Brain Research (MPIBR) and Biophysics (MPIBP) to develop custom mass spectrometry-based solutions for research questions raised at both institutes. The MPIBP focuses on structures and mechanisms of membrane proteins, and makes use of x-ray crystallography and electron cryo microscopy and tomography to gain new insights into their structure and function. Research at the MPIBR is mainly focused on Connectomics, neural systems and synaptic plasticity in different model systems. The developed methods range from individual membrane protein characterization and analysis of post-translational modifications, Hydro-gen-Deuterium exchange mass spectrometry and functional assays on membrane protein complexes to quantitative techniques including (pulsed) SILAC and Label-Free Quantitation, miniaturized setups (PALM-excised tissue samples) and mass spectrometry (MS) Imaging.

Internal work

Dr. Langer’s laboratory plays an interactive role between the MPI for Brain Research and the MPI for Biophysics: he receives funding from both institutes to conduct research and design MS assays which address questions raised on both sides. The laboratory currently supports four PhD students, one master student, one engineer and one technician. The engineer is responsible for running the equipment in the laboratory, and the technician for sample preparation and biochemistry work. Each student has her/his own research projects as well as joint projects in both institutes.

Researching molecular membrane biology

The key function of biological membranes – the structure surrounding cells and intracellular compartments – is to protect the cell and regulate the entry and exit of specific molecules. Membrane proteins are incorporated into the lipid bilayer which makes up the membrane structure, enabling the specific passage or transport of

Dr. Julian Langer’s laboratory at the Max Planck Institutes of Biophysics and Brain Research uses cutting-edge MALDI mass spectrometry technology to support their research in molecular membrane biology.

“The instruments from Bruker go above and beyond what is currently available in the field – we are working with

specialists at the highest level to bring forward novel solutions to questions in membrane proteomics.”

selected substances across the membrane. Cell communication is another vital role of membrane proteins: many act as sensors and receptors, to receive incoming signals at the outer surface of the membrane and transduce across the bilayer. Membrane proteins serve key cellular functions in all cells, ranging from substrate sensing in prokaryotes to voltage-gated ion channels in nerve cells in the human brain.

Membrane mass spectrometry

The goal of the Department of Molecular Membrane Biology headed by Prof. Hartmut Michel is to understand the precise mechanism of both transportation and signalling actions of mem-brane proteins. A comprehensive understanding is only possible with an accurate knowledge of the protein’s atomic structure – commonly obtained using X-ray crystallography or electron microscopy and tomography – and subsequently, function and mechanism is deduced.

The lab of Julian Langer contributes mass spectro- metry-based, complementary tools to study every component of biological membranes, by performing qualitative and quantitative proteomics analyses to characterise membrane proteins, identify and characterise ligands, perform functional studies on membrane proteins, and recently, for lipidomics analyses. Matrix-Assisted Laser Desorption/ Ionisation (MALDI)-MS-based assays are also used for the analysis of individual membrane protein crystals, a technique that allows direct evaluation of the proteins present in the crystals prior to the time - and cost - intensive process of diffraction data acquisition and evaluation. Dr. Langer describes this important component of his laboratory’s work with the MPI for Biophysics:

“We have recently begun looking at protein modifications, particularly for small subunits in protein complexes. Mass spectrometry is a complementary tool to X-ray crystallography and electron microscopy (EM), to identify and characterise these protein complexes. Despite the improvements over the past 5-10 years in the speed at which EM structures can be obtained, and the resolution that can be recorded, we still get surprises in the form of new, previously uncharacterized subunits. With MS, we can now unambiguously identify and assign the proteins and modifications present in these structures.”

Introducing mass spectrometry to the MPIs

The first MS instrument – Bruker’s microTOF Q2 – was installed in the MPI laboratory in 2009, when Dr. Langer joined the MPI for Biophysics as a post-doctorate and then later started his laboratory in Prof. Michel’s department. Dr. Langer describes the early use of MS in the laboratory when he first started working with Professor Michel, during a study on a Pseudomonas oxidase complex, which proved its validity:

“I was given a sample in which to routinely analyse the protein complexes. I found proteins from our target strain, but also from a different strain - and another isoform of our target protein. It turned out there had been a mistake sequencing the organism – there had been a primer jump – and the protein I identified which looked to be from a different organism was actually from the target strain. We re-sequenced the operon and got the right sequence for that protein, and subsequently solved the structure [1].”

Using Peptide Mass Fingerprinting (PMF), the laboratory employed proteolytic digests of the target proteins to identify them by matching the acquired tandem-mass spectra to a proteome database. It developed optimised protocols for the analysis of membrane proteins, enabling the acquisition of sequence coverage also of hydrophobic domains in membrane proteins.

In 2010, Dr. Langer introduced Bruker’s autoflex III Smartbeam MALDI-MS into the laboratory for functional assays, in order to rapidly screen samples and record full length mass spectra from samples which did not ionise well with an electrospray source. The laboratory has been able to publish a number of functional assays using the autoflex.

In 2012, the joint laboratory between the two MPIs was established as a collaborative center. Dr. Langer obtained funding to purchase additional mass spectrometers also for shotgun proteomics to supplement the existing ultra-high resolution QTOF, the maXis. In 2015, Julian Langer’s laboratory started to use Hydrogen Deuterium Exchange (HDX) coupled with MS to gain further insights into ligand binding and fully track conformational dynamics. Further to this, in 2016, Dr. Langer extended the laboratory’s MS portfolio by purchasing Bruker’s rapifleX MALDI-TOF/TOF system and an Impact II ESI-Q-TOF coupled to a nanoElute UPLC.

“We realised that we had so many protein structures with unknown densities, we needed a tool to directly sequence them,” explains Dr. Langer, commenting: “At that point I had already been working with someone from Bruker for three or four years – she showed me some really interesting data on new subunits of oxidase complexes using the ultrafleXtreme. The rapifleX was not even on the market at this point, so I went to Bremen and looked at a couple of spectra and from then on, I knew I wanted the rapifleX. I received a grant for the machine and realised that for many of the brain research questions we wanted to address, MALDI imaging would also be an interesting addition to the portfolio.”

The rapifleX is primarily used for the sequencing of unknown densities where good cleavage sites, for example, are not available for classical bottom-up proteomics. The laboratory can obtain direct top-down proteomics data by MS/MS sequencing of these species [2]. In addition, one of Dr. Langer’s students is working solely on MS-Imaging, which is mainly used by the MPI for Brain Research. Dr. Langer describes how his laboratory has progressed since the purchase of the rapifleX:

“Last year (2016) we also bought an impact II™ ultra-high resolution QTOF mass

spectrometer, because we also want to use MS in the context of an ImageID workflow, together with a rapifleX for imaging. This is something that is still

currently in development, but the initial results yielding

parallel localizations for hundreds of proteins are very

promising.”

Collaborations

As one of the industry’s global academic experts, Dr. Langer’s direct contact with mass spectrometry specialists is invaluable. Dr. Langer describes his relationship with Bruker:“I’ve been very close with Bruker from the beginning, from being one of the first laboratories in the world to have a maXis. I have direct contact with the specialists in Bremen who provide a service which is above what is typical in the industry. For example, when we were conducting complex research on a new drug designed against Mycobacteria, I travelled to Bremen for a couple of days and we talked almost daily until everything was up and running. We were lucky enough to have a prototype rapifleX and thus had tremendous service support onsite. We’re collaborating with Bruker to further develop this machine: at the moment we can achieve high mass accuracy and resolution for an MS/MS spectrum of a 8700 m / z species, which is something no other machine can do at the moment except the one in Bremen and the one in my laboratory in Frankfurt.”

Global partnerships

In addition to working with Bruker to develop sophisticated mass spectrometry techniques, the laboratory at the Department of Molecular

Membrane Biology collaborates with institutions across the world to solve some of the field’s most challenging problems.

“We have quite a few external collaborations” comments Dr. Langer, continuing: “for instance, right now we are investigating the proteomes of alkaliphilic bacteria with Terry Krulwich’s group in New York, with the lab of Xabi Contreras in Bilbao we study protein-lipid interactions and with Thomas Meyer’s group at Imperial College, London, we work on mycobacterial drug targets. For the most part, people approach me with requests for partnership, so I have to be very selective with the collaborations I pick up. Because I have a small lab, time is our most precious commodity – both measurement and personal time. We can engage only in a small fraction of the collaborations we are asked to do from external groups, but I always try to establish contact with colleagues that can help if we are unable to do so ourselves.”

In 2017, Dr. Langer received funding as part of a special priority programme (SPP 2002) by the German Research Foundation. The research

focus of the program lies in the identification and characterization of small prokaryotic proteins that often play important regulatory and functional roles but have not been characterized due to their biochemical properties. They are often low abundant, heavily modified or processed and not accessible to conventional bottom-up proteomics due to missing cleavage sites.

“We believe that the rapifleX-based direct sequencing will

prove extremely valuable for characterising these

polypeptides.

Using the TOF/TOF high mass module, we do not require proteolytic digests, and can acquire direct sequence information up to 10 kDa, and also directly see if the polypeptide has been modified.”

Past and current work

The laboratory carries out a combination of routine and specialist activities in its daily running. Approximately 20-30% of run time is for routine samples, depending on the instrument. Almost

no routine samples are run using HDX, but with the maXis it is almost 80-90% of run time, where simple identification of proteins present in a sample that has been expressed for X-ray crystallography or electron cryo tomography/microscopy is required, for example.

Current work being undertaken at Dr. Langer’s laboratory spans a number of different organisms, with work in Biophysics focussing mainly on prokaryotic targets. Recent studies centered on the characterization of respiratory chain complexes that included:

• Cbb3 cytochrome c oxidase in Pseudomonas stutzeri

• Bd quinol oxidase in Geobacillus thermodenitrificans

• ATP Synthase in Mycobacterium phlei as a drug target

Cbb3 cytochrome c oxidase

Pseudomonas are soil bacteria and opportunistic pathogens to humans, primarily affecting immunocompromised patients. Dr. Langer’s lab contributed to solving the crystal structure for the cbb3 cytochrome c oxidase from Pseudomonas stutzeri – a protein complex involved in the bacterial respiratory chain – in 2010 [3]. However, at the time, a single-transmembrane-helix subunit in the structure could not be identified. Using direct sequencing, the red helix (see Figure 1, representing an identity which was previously unassigned) was recently identified and shown to play an essential role in anaerobic respiration. Dr. Langer explains the laboratory’s findings:

“We realised we needed a tool allowing us to identify proteins which cannot otherwise be identified using conventional bottom-up or top-down proteomics approaches. We had tried to identify the helix with 17 different combinations of proteases, and it didn’t fly with any of the top-down electrospray experiments. The helix is about 4 kDa, so at that point, we were not able to acquire an information-rich MS2spectrum of this polypeptide on our mass spectrometers. Using a customized ultrafleXtreme at Bruker and then later our rapifleX, we managed to obtain the spectra and identify the polypeptide.”

Bd quinol oxidase

A second study involving a previously unidentified subunit focused on the bd quinol oxidase in Geobacillus thermodenitrificans, an aerobic thermophilic bacterium found in extreme environ-ments. The bd quinol oxidase is also a promising drug target in mycobacteria, so the laboratory’s work on revealing the structure will have important implications in future research. The laboratory used the rapifleX to directly sequence the unde-scribed subunit, and obtained a mass accuracy of below 0.01 Daltons across the entire mass range [2]. The direct MS2 spectra of the single transmembrane helices can be seen in Figure 2. Dr. Langer explains the importance of powerful instrumentation in his laboratory’s work:

“What we can accomplish with the rapifleX MALDI-TOF/

TOF is really outstanding, and we are very grateful for the dedication of the Bruker team who assist us with our

research goals.

At the moment, there is no alternative to the rapifleX in terms of resolution and mass accuracy for high m/z species, so this is where Bruker really shines.”

Figure 1. Identification and characterization of a novel µ-protein subunit in a prokaryotic terminal oxidase. A Structure of the cbb3 cytochrome c oxidase from P. stutzeri (pdb: 3mk7). Subunits CcoN, CcoP and CcoO are colored in dark grey, novel µ-protein subunit CcoM in red. Grey bars indicate membrane borders. B Annotated MALDI-MS/MS spectrum of purified CcoM (Mascot score: 294). C CcoM amino acid sequence fitted into 3mk7 electron densities of a preliminary poly-alanine model (2Fobs–Fcalc: 1.5 sigma, colored in blue; Fobs–Fcalc: 3.0 sigma, colored in green). D Growth curves of P. stutzeri wild type (black curve) and the ΔCcoM variant (red curve) in anaerobic conditions

A B

C D

Mycobacteria and bedaquiline

The laboratory’s investigation into mycobacterial membrane proteins is at the forefront of antibiotic research. In 2015, the lab of Thomas Meier solved the structure of the target protein of the antibiotic bedaquiline, used as treatment against multi-drug resistant tuberculosis. Dr. Langer contributed to this project by providing essential tools required for screening crystals, and a MALDI-MS-based competition assay that allows screening of antibiotics and variants with point mutations leading to drug resistance. He describes how his laboratory’s work on the target protein has changed the way how new bedaquiline-derived drugs for tuberculosis can be tested:

“With our findings published in 2015, we now know exactly how the antibiotic bedaquiline works, but we also know that individual point mutations in that protein are going to render bedaquiline ineffective. Now this is the first new antibiotic after 40 years of research on mycobacteria, and it reduces treatment time from 6 months to approximately 6 weeks. But we know we are on borrowed time. So we have a head start, but we need fast and reliable tools to screen both derivatives that are still active against bedaquiline-resistant variants, and we need a tool to directly diagnose point mutations in vivo.”

Figure 2: MS2 spectrum of CydS, a novel bd quinol oxidase subunit

Figure 3: X-ray structure of the mycobacterial c-ring (green) with bound bedaquiline (black)

Figure 4: Competition assay of bedaquiline and DCCD based on MALDI-MS, allowing direct monitoring of drug binding to the target protein’s active site and evaluate activity. Increasing concentrations of bedaquiline (colored as indicated in the legend) show decreasing amounts of DCCD labeling, indicating direct competition of both compounds for the same binding site

Brain Research: Artificial amino acids and MS Imaging

Through the MPI for Brain Research, Dr. Langer’s laboratory has focused on implementing, optimizing and further developing different proteomics applications that mainly focus on complex samples. In addition to proteome turnover studies and sub-proteome profiling of purified cellular preparations, his laboratory specializes in the use of artificial amino acids to selectively label newly-synthesized proteins. This technique, pioneered by Erin Schuman, Director at the MPI for Brain Research, allows purification and quanti-fication of the cellular response to external stimuli - overcoming the challenge of “finding the needle in the haystack” in conventional shotgun proteomics where the constitutively expressed proteome normally creates an extremely challen- ging background. Together with the Schuman lab, Dr. Langer published the most comprehensive proteome dataset on homeostatic scaling in primary hippocampal neurons in 2016 (Schanzenbächer et al., Neuron, 2016). Recently, the Schuman lab generated a transgenic mouse that allowed cell-type specific labeling of cellular proteomes in vivo, for example for excitatory neurons in the hippocampus and inhibitory neurons in the cerebellum. The work was recently published in Nature Biotechnology [7].

In follow-up experiments, the laboratory plans to use MALDI Imaging to make use of cell-type specific labelling with artificial amino acids, and tag cells to visualise the local lipid and small molecule composition at the location where the artificial amino acid is incorporated into proteins. Dr. Langer describes this work:

“For me, this is one of the most interesting brain research projects we’re

currently undertaking, partly because this is something really new which nobody

else has done before. We are using the rapifleX for preliminary imaging

experiments in this context.”

For the enriched environment and field conditioning experiments in mice, the changes in the brain caused by learning activities are almost imperceptible, so both high resolution and high speed instrumentation is vital to identify and monitor nuances in behaviour in different stimulation environments.

Figure 5: MALDI imaging of tissue sections. Panel A shows a mouse brain imaged using MSI and visualized using distributions of four different m/z species in a sagittal section (lipid imaging). Panel B shows a transversal section through a zebrafish brain, with six different m/z species plotted and superimposed with an optical image. Panel C shows a sagittal section through a rat brain imaged in the m/z range 2000-20000, plotting small protein distributions as indicated in the figure. Lower panels show superimposition of m/z species enriched in the pyramidal cell layer in the hippocampus (left) and the ependyma (right)

A

B

C

“If we didn’t have a laser with the speed of the rapifleX, we couldn’t undertake the

research we want to. This is a very clear selling point for the

instrument”

explains Dr. Langer, continuing: “This type of analysis requires screening of a minimum of 10-20 animals per condition, with 20-30 brain slices are taken from each. Using traditional methods, it would take years to record all the samples we wanted to analyse, which is a time-frame we cannot afford.”

Membrane proteomics in the future

The recent developments in mass spectrometry technology have enabled front-line laboratories across the world, such as that at the Max Planck

Institutes, to propel their research into new spheres. With a laboratory equipped with the highest specification instruments, Dr. Langer is pushing the capabilities of what can be measured in brain research:

“Before I started my laboratory and begun working with the MPI for Brain Research and the MPI for Biophysics, there were no mass spectrometers at the institutes. I have built up the capabilities of the laboratory and made particular use of the growing knowledge offered by the MS industry to allow the laboratory to provide greater and stronger services to the two institutes” comments Dr. Langer.

For more information on the mass spectrometry facilities at the Max Planck Institutes, please visit http://www.biophys.mpg.de/en/ms-facility.html.

For more information on the rapifleX, please visit https://www.bruker.com/products/mass-spec-trometry-and-separations/maldi-toftof/rapiflex/overview.html

References

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

[2]. Safarian, S., Rajendran, C., Muller, H., Preu, J., Langer, J.D., Ovchinnikov, S., Hirose, T., Kusumoto, T., Sakamoto, J. and Michel, H. (2016) Structure of a bd oxidase indicated similar mechanisms for membrane integrated oxygen reductases, Science, 352(6285): 583-586.

[3]. Kohlstaedt, M., Buschmann, S., Xie, H., Resemann, A., Warkentin, E., Langer, J. D. and Michel, H. (2016) Identification and characterization of the novel subunit CcoM in the cbb3-Cytochrome c Oxidase from Pseudomonas stutzeri ZoBell, American Society for Microbiology, 7(1):e01912-15.

[4]. Preiss L, Langer JD, Yildiz Ö, Eckhardt-Strelau L, Guillemont JE, Koul A, Meier T. (2015) Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline, Science Advances, 1(4):e1500106.

[5]. Bausewein T, Mills DJ, Langer JD, Nitschke B, Nussberger S, Kühlbrandt W. (2017) Cryo-EM Structure of the TOM Core Complex from Neurospora crassa, Cell, 170(4):693-700.e7.

[6]. Schanzenbächer CT, Sambandan S, Langer JD, Schuman EM. (2016) Nascent Proteome Remodeling following Homeostatic Scaling at Hippocampal Synapses, Neuron, 92(2):358-371.

[7]. Alvarez-Castelao B, Schanzenbächer CT, Hanus C, Glock C, Tom Dieck S, Dörrbaum AR, Bartnik I, Nassim-Assir B, Ciirdaeva E, Mueller A, Dieterich DC, Tirrell DA, Langer JD, Schuman EM. (2017) Cell-type-specific metabolic labeling of nascent proteomes in vivo, Nature Biotechnology 35, 1196–1201.

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Customer InsightsFrom Individual Membrane Protein Complexes to in vivo Proteome Dynamics: Developing New Mass Spectrometry Solutions

High speed MALDI mass spectrometry instrumentation is an essential element in multidisciplinary proteomics research at the Max Planck Institutes for Biophysics and Brain Research