Nanoelectrospray ionization emitters with submicron tip diameters have significant advantages for use in native mass spectrometry, including the ability to produce resolved charge-state distributions for proteins and macromolecular complexes from standard biochemical buffers that contain high concentrations of nonvolatile salts and to prevent nonspecific aggregation that can occur during droplet evaporation. We report on various factors affecting the tip morphology and provide suggestions for producing and using emitters with submicron tips. Effects of pulling parameters for a Sutter Instrument P-87 tip puller on the resulting tip diameter and morphology are shown. The "Pull" parameter has the largest effect on tip diameter, followed by "Velocity", "Pressure", and "Heat", whereas the "Time" parameter has minimal effect beyond a lower threshold. High "Pull" values generate emitters with multiple tapers, whereas high "Velocity" values generate a tip with only a single tapered region. A protocol for producing reproducible emitters in the submicron size range, as well as guidelines and tips for using these emitters with standard biochemical buffers that contain high concentrations of nonvolatile salts, is presented with the aim of expanding their use within the native mass spectrometry community.
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