Assessment of Sub-50 nm Nanopipette Emitters for Native Electrospray Ionization Mass Spectrometry

Abstract

Native mass spectrometry (nMS) is well established for measuring protein masses and stoichiometries using nanoelectrospray ionization (nESI), yet salt adduction and source activation energies can limit routine measurements. In this study, we benchmark submicron quartz nanopipette nESI emitters (<50 nm internal diameter) across four mass spectrometry platforms (quadrupole-time-of-flight, quadrupole-Orbitrap, tribrid-Orbitrap platforms and timsTOF) and a wide protein range (14.5–800 kDa). We analyzed the intrinsically disordered protein alpha synuclein (αS; 14.5 kDa) and holo-myoglobin (17 kDa) with myoglobin further characterized over a range of concentrations (10 μM-1 nM) and capillary voltages to determine concentration ranges and define a gentle operating regime. We additionally observed reduced Na+ adduction and preservation of the Zn2+-bound metalloproteoform of carbonic anhydrase II (29 kDa). Proteins and protein complexes spanning the mid-to-high mass range including ovalbumin (∼44 kDa), malate dehydrogenase (∼70 kDa), transferrin (80 kDa), glutamate dehydrogenase (∼350 kDa), β-galactosidase (∼465 kDa), and GroEL (∼800 kDa) were readily detected using nanopipette emitters. Compared with conventional 1–2 μm internal diameter borosilicate emitters, quartz nanopipettes provided higher signal-to-noise ratios and fewer adducts. Finally, direct analysis of a clarified bacterial lysate expressing αS yielded a clear monomeric charge-state distribution, demonstrating compatibility with complex biological matrices. Collectively, these results establish quartz nanopipette nESI as an instrument-portable, salt-tolerant approach suitable for routine nMS analysis across a broad range of protein molecular weights and sample complexities.