Analytical Methods

High resolution mass spectrometry for intact protein analysis

Project INTACT
Coordinator: C. Masselon
Funding: OSEO/FRM

In conventional proteomics, chromatography and mass spectrometry are used in combination to separate mixtures of proteolytic peptides obtained after digestion of the proteins of interest. However, the molecular actors of life are the proteins, and the information derived from their digestion products is unavoidably fragmentary. For example, the characterization of alternative splice isoforms, combinations of post-translational modifications, or endogenous protein processing are extremely difficult, when not totally impossible to study using conventional approaches. Consequently, to complement existing proteomics methods, new technologies termed “top-down” proteomics have emerged recently. Top down proteomics consist in mass spectrometric analysis of whole domains, proteins, or sub-complexes using specific separation and fragmentation techniques to determine their primary sequences, and the nature and position of post-translational modifications. As a matter of fact, the protein’s mass includes its modifications state, possible truncation; disulfide bridges, and even, under controlled conditions, the masses of its cofactors such as hemes or metal ions. Through research programs funded by OSEO and FRM, we are developing, in collaboration with industrial (PX Therapeutics, Prediction Biosciences) and academic (IBS, iRTSV) partners the instrumental and methodological infrastructures to address these questions for purified proteins or complexes.

New concept for ultrahigh molecular mass measurements: nano-electromechanical mass sensors

Project NEMS-MS
Coordinator: S. Hentz, LETI
EDyP correspondent: C. Masselon
Funding: ANR ASTRID 

Gravimetric devices permit mass measurements in the macro-world (kilograms to nanograms), whereas mass spectrometers perform mass-to-charge ratio measurements on the infinitesimal scale (attograms to yoctograms). In between these two regimes, lies the “meso-world” where viruses, spores, fungi, bacteria, and cellular organelles roam. Owing to a lack of appropriate technology, these species have escaped mass measurement until now. Nano-Electro-Mechanical Systems (NEMS) are extremely sensitive to inertial mass of both neutral and ionized individual particles accreting upon their surface, and have unprecedented resolving power in the aforementioned mass range. While proofs of concept of the NEMS-MS principle have been demonstrated (Nature Nanotechnology 2012, 7:602–608), they were performed with cumbersome and low efficiency interfaces that preclude the efficient utilization of these devices to systematically explore the mass range of interest. In collaboration with LETI, Caltech, University of Washington, and Université Pierre et Marie Curie, we are developing new delivery vehicles for NEMS, the only devices capable of performing mass measurements in the meso-world regime.

Surface acoustic wave nebulization of biomolecules for mass spectrometry.

Project SAWN
Coordinator: C. Masselon
Funding: CEA Eurotalent

Over the past two decades, mass spectrometry (MS) has become an essential tool in modern biology. This would have been impossible without soft ionization methods able to bring non-volatile, thermally labile bio-molecule into the gas phase for further manipulation by electric and magnetic fields. The most prominent soft ionization methods are electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI). While these methods constitute the core of current bio-MS technologies, they are not totally devoid of limitations. For instance, ESI is salt-sensitive and its efficiency depends on the quality of the spraying needle, while MALDI is afflicted by noise at low m/z due to matrix ions. Therefore, the search for improved soft-ionization methods is continuing and several so-called ambient desorption/ionization methods have been proposed. (eg. LESI, DART) Acoustic waves can be generated at the surface of a piezzo-electric material by means of an inter-digitated transducer activated by a radio-frequency (RF) signal. Depending on the power of the applied waveform, acoustic waves have the capacity to create convection in a liquid, eject or even entirely nebulize a droplet. This last capability, termed surface acoustic wave nebulization (SAWN), has recently been used to generate a mist from a droplet of solution that was analysed in a mass spectrometer, and showed the formation of labile molecular ions (Anal Chem. 2010 82(10): 3985–3989). This method was later shown to impart less energy to the analyte than even ESI (J Am Soc Mass Spectrom. 2012 23(6):1062-70). In cooperation with the start-up company Deurion and University of Washington, we are working to develop applications for SAWN, particularly for high mass compounds such as proteins and complexes.