Abstract
Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.Graphical ᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-016-1381-5) contains supplementary material, which is available to authorized users.
Highlights
Mass spectrometry (MS) of intact membrane proteins and their complexes has developed rapidly over the past 10 years [1]
We have shown that membrane proteins experience similar charge reduction effects to soluble proteins when analyzed under negative polarity electrospray ionization (ESI)
No immediate advantages to using negative mode ESI for soluble proteins exist [12], we have highlighted some key advantages in the case of membrane proteins without compromise in sensitivity
Summary
Mass spectrometry (MS) of intact membrane proteins and their complexes has developed rapidly over the past 10 years [1]. New approaches are needed to provide opportunity for MS studies of a greater range of membrane proteins. Whilst Brian’s scientific achievements are undisputed, perhaps less appreciated is his ability to motivate and mentor his group members and colleagues in mass spectrometry. Brian encourages others to pursue new directions, to find novel avenues, and to retain focus on the biological question as the driving force of the investigation. It is this philosophy that has led to his numerous scientific breakthroughs and will hopefully inspire others to follow in his footsteps
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