Study on Manufacturing Parameters of Submicron Emitters for Native Mass Spectrometry

RATIONALE: Submicron nanoelectrospray emitters can greatly enhance the performance of native mass spectrometry (nMS) by increasing ionization efficiency and improving salt tolerance. This enhancement enables more accurate determination of intact protein molecular weights under near-physiological conditions and minimizes nonspecific aggregation during ionization. However, the high cost of these emitters limits their widespread use in nMS. In contrast, homemade emitters offer a cost-effective alternative that can significantly reduce the overall expense of native mass spectrometry. METHODS: The impact of manufacturing parameters—loop, heating power, pulling force, cooling time, and hard pull trigger speed—on the morphology of submicron nanoelectrospray emitters during the high-temperature pulling process was systematically investigated. Subsequently, the performance of the self-prepared emitters was evaluated by characterizing three representative proteins with varying molecular weights. RESULTS: Heating power and pulling force were identified as key factors controlling the tip inner diameter (ID) and cone length. By employing multiple-loop processing to reduce the pulling force, emitters suitable for nMS were achieved. The tip ID was successfully reduced from 3 μm to 800 nm, which enhanced salt tolerance—BSA from 300 to 800 μM, cytochrome C from 800 to 2000 μM, and IgG from 10 to 50 μM—as well as detection sensitivity—BSA from 0.1 to 0.06 μM, cytochrome C from 1.5 to 0.25 nM, and IgG from 0.7 to 0.1 μM—while also reducing nonspecific protein aggregation.Conclusion: The fabricated submicron nanoelectrospray emitters are ideally suited for native mass spectrometry (nMS) analysis. By elucidating the relationship between pulling parameters and emitter morphology, it becomes straightforward to produce emitters with diverse tip IDs and specifications, thereby advancing the development of nMS.