Modulating proteoform charge states and signal intensities in LC-MS using a nanoflow sheath liquid interface.

To investigate intact proteins in biological samples, hyphenation of mass spectrometers (MS) with separation techniques like liquid chromatography (LC) or capillary zone electrophoresis (CZE) is crucial. While reversed-phase nanoLC is typically coupled directly ("sheathless") to a mass spectrometer, for CZE-MS coupling, sheath liquid interfaces are typically used. Here, we coupled the reversed-phase nanoLC to an Orbitrap Fusion Lumos using a nanoflow sheath liquid interface (nanoCEasy-interface) and compared the results with the conventional ("sheathless") coupling. We investigated the influence of emitter orifice diameter and the use of sheath liquid on proteoform ionization using constant LC flow independently of any high voltage as required for CZE separations. We found that a smaller emitter orifice is beneficial regarding proteoform intensity. Furthermore, the used sheath liquid also has a major effect on the resulting proteoform intensity, especially for emitters with a larger orifice and thus increased dilution of the column's effluent with sheath liquid. Additionally, the sheath liquid influenced the charge state distributions of proteoforms, e.g., shifting the most abundant charge state of histone H4 from 15+ to 8+ upon addition of a 2-propanol:water (1:1, v/v) with 0.5% formic acid sheath liquid to the column effluent. This approach allows for easily reducing co-isolation of proteoforms by shifting their charge states to higher m/z and thus increasing the m/z difference between proteoforms with similar mass.