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

Abstract

Abstract Native mass spectrometry (nMS) is well established for measuring protein masses and stoichiometries using nano-electrospray 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 three mass spectrometry platforms (quadrupole-time-of-flight, quadrupole-Orbitrap, and tribrid-Orbitrap platforms) and a wide protein range (14.5-800 kDa). We analysed the intrinsically disordered protein alpha synuclein (αS; 14.5 kDa) and holo-myoglobin (17 kDa) over a range of concentrations (10 μM-1 nM) and capillary voltages to determine limits of detection 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 clarified bacterial lysate expressing α-synuclein 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.

The authors' abstract, as published at the source. Journal of the American Society for Mass Spectrometry, 2026 · DOI ↗