Abstract
High-field asymmetric waveform ion mobility spectrometry (FAIMS) enables the separation of ions on the basis of their differential mobility in an asymmetric oscillating electric field. We, and others, have previously demonstrated the benefits of FAIMS for the analysis of peptides and denatured proteins. To date, FAIMS has not been integrated with native mass spectrometry of folded proteins and protein complexes, largely due to concerns over the heating effects associated with the high electric fields employed. Here, we demonstrate the newly introduced cylindrical FAIMS Pro device coupled with an Orbitrap Eclipse enables analysis of intact protein assemblies up to 147 kDa. No evidence for dissociation was detected suggesting that any field heating is insufficient to disrupt the noncovalent interactions governing these assemblies. Moreover, the FAIMS device was integrated into native liquid extraction surface analysis (LESA) MS of protein assemblies directly from thin tissue sections. Intact tetrameric hemoglobin (64 kDa) and trimeric reactive intermediate deiminase A (RidA, 43 kDa) were detected. Improvements in signal-to-noise of between 1.5× and 12× were observed for these protein assemblies on integration of FAIMS.
Highlights
field asymmetric waveform ion mobility spectrometry (FAIMS) for the analysis of peptides and denatured proteins
Much of our current interest in FAIMS derives from the improved S/N of protein signals detected following direct sampling of biological substrates, including dried blood spots, bacterial colonies, and tissue.[17−21] In those works, FAIMS mass spectrometry (MS) was coupled with liquid extraction surface analysis (LESA) using denaturing solvents, i.e., the proteins detected were intact but unfolded
The optimum compensation voltage (CV) for transmission was determined by stepping through the CV range −60 V to −10 V at a rate of 1 V per scan
Summary
Samples of carbonic anhydrase (CAH), concanavalin A (ConA), and alcohol dehydrogenase (ADH) in ammonium acetate solution were analyzed by direct infusion nESI FAIMS mass spectrometry. Information) of the mouse kidney section, which is characterized by abundant vasculature, was sampled using ammonium acetate solution (200 mM) + 5% MeOH These sampling conditions are known to be suitable for detection of the hemoglobin heterotetramer (αβ2H)[2] (PDB entry 3hrw).[25,27] Intact hemoglobin tetramer (αβ2H)[2] (63.6 kDa) ions were observed, i.e., were successfully transmitted by the FAIMS device. LESA sampling of the rat kidney tissue was performed in the cortex region (see Figure S4b, Supporting Information) using an extraction solvent comprising ammonium acetate solution (10 mM) containing 0.125% C8E4 detergent This combination of sampling conditions and location has previously been shown to result in the detection of ions corresponding to the homotrimer reactive intermediate deiminase A (RidA).[26] The homotrimer ions of RidA (42.6 kDa, PDB entry 1qah) were successfully transmitted through the FAIMS device and observed in charge states 9+ through 12+ (protein assignment was based on mass measurement).
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