Protonation Isomers of Highly Charged Protein Ions Can Be Separated in FAIMS-MS

High-field asymmetric waveform ion mobility spectrometry-mass spectrometry (FAIMS-MS) can resolve over an order of magnitude more conformers for a given protein ion than alternative methods. Such an expansion in separation space results, in part, from protein ions with masses of \u3e29 kDa undergoing dipole alignment in the high electric field of FAIMS, and the resolution of ions that adopt pendular vs free rotor states. In this study, FAIMS-MS, collision-induced dissociation (CID), and travelling wave (TW) IMS-MS were used to investigate the pendular and free rotor states of protonated carbonic anhydrase II (CAII, 29 kDa). The electrospray ionization additive 1,2-butylene carbonate was used to increase protein charge states and ensure extended ion conformations were formed. For relatively high charge states in which dipole alignment occurs (30e38þ), FAIMS-MS can baseline resolve the isobaric pendular and free rotor ion populations. For TWIMS-MS, these same charge states resulted in monomodal arrival time distributions with collision cross sections corresponding to highly extended ion conformations. Interestingly, CID of FAIMS-selected pendular and free rotor ion populations resulted in significantly different frag-mentation patterns. For example, CID of the dipole aligned CAII 37þ resulted in cleavages C-terminal to residue 183, 192 and 196, whereas cleavage sites for the free rotor population occurred near residues 12 and 238. Given that the cleavage sites are ’directed’ by protonation sites in the CID of protein ions, and highly charged protein ions adopt extended conformations with the same or very similar collision cross sections, these results indicate that the pendular and free rotor populations separated in FAIMS can be attributed to protonation isomers. Moreover, the extent of protein ion charging in FAIMS-MS decreased substantially as the carrier gas flow rate decreased, indicating that ion charging in FAIMS-MS can be limited by proton-transfer reactions. Given that the total mass of proton charge carriers corresponds to less than 0.2% the mass of CAII, we anticipate that FAIMS-MS can be used to separate intact isobaric proteoforms with masses of at least ~29 kDa that result from alternative sites of post-translational modifications

Cold vapor integrated quartz crystal microbalance (CV-QCM) based detection of mercury ions with gold nanostructures

In this study, we developed a novel method of integrating the well-accepted cold vapor technique with gold nanostructured based quartz crystal microbalance (QCM) devices to selectively detect mercury ions (Hg2+). This method allows for the conversion of aqueous mercury ions into elemental mercury (Hg°) vapor form and thereon use the highly sensitive QCM based mercury vapor sensors to detect the evolved mercury. The method involves reducing mercury chloride (HgCl2) in contaminated water by mixing it with a 2% tin chloride (SnCl2) solution in order to evolve Hg° vapor from the liquid mixture. The selectivity and sensitivity performance of each gold nanostructure, namely, the control Au thin film (Au-ctrl), Au-nanospheres (Au-NS) and Au-nanourchins (Au-NU), towards mercury vapor was evaluated. It was found that Au-NS and Au-NU sensors displayed up to 79% and 243% higher response magnitudes than the Au-ctrl sensor for various concentrations of HgCl2, respectively. All three sensors exhibited repeatable sensing performance when reporting the concentrations from 5 sensing events involving 500 ppb HgCl2 solution with Au-ctrl, Au-NS and Au-NU having the coefficient of variance (CoV) values of ˜5.7, 2.9 and 3.8%, respectively. Moreover, the sensors were observed to operate in the linear region with the mercury ion concentration range calibrated and tested. Importantly, the sensors showed no cross-interference effects when tested toward Hg2+ ions with and without the presence of other metal ions such as lead, cadmium, manganese, iron, and zinc. The results indicate that the CV-QCM technique developed is feasible to be potentially used in real-world mercury monitoring applications