We incorporate nanoelectromechanical systems (NEMS) into a state-of-the-art commercial mass spectrometer (Q Exactive Plus with Orbitrap detection). This unique hybrid instrument is capable of ionizing molecules up to 4.5 MDa in their intact native state, isolating molecules of interest according to their mass-to-charge ratio, performing high resolution mass spectrometry (MS), and delivering those molecules to the NEMS. We use NEMS optimized for detecting the inertial mass of adsorbed species directly, which contrasts with indirect measurements of the mass-to-charge ratio performed with typical instruments. This unique form of mass spectrometry, NEMS-MS, with its single-molecule sensitivity, has promising applications to the fields of proteomics and native mass spectrometry, including deep proteomic profiling, single-cell proteomics, mass spectrometry-based imaging, or identifying viruses in their in vivo state.
We analyze intact E. coli GroEL chaperonin, a noncovalent 801 kDa complex consisting of 14 identical subunits. GroEL was sent to NEMS operated with the first two vibrational modes monitored in real time. Molecules physisorbing to the NEMS cause an abrupt shift in its resonance frequencies. The change in resonance frequencies is used to calculate the mass of each molecule. A mass spectrum is compiled with a main peak of 846 kDa, close to the expected value, and a secondary peak resolved near twice the mass of GroEL.
Measurements are then performed operating the first three modes simultaneously. Using a technique called inertial imaging, frequency shifts are used to calculate the first three mass moments: mass, position, and variance (size). This is used to distinguish between adsorbates arriving in a single, point-like distribution or a more extended distribution, thus demonstrating a rudimentary form of molecular imaging.
Two new theories are presented for analyzing frequency-shift data. The first approach offers a more streamlined approach for calculating the mass moments. This approach is used to improve the mass spectrum of the GroEL calculated using three-mode data, producing a main peak almost fully resolved at 805 kDa. An entirely different approach is presented that allows for obtaining the mass density distribution of an adsorbed molecule (i.e., imaging) with a higher number of modes.</p
