Table 1. Troubleshooting Table.

Step	Problem	Possible reason	Possible solution
3	The protein is unstable, as detected by monitoring the SEC profile or observing precipitates.	Certain proteins are stable only in specific detergents.	Screen different detergent to determine a list of detergent for the protein of interest to achieve the correct balance between stability and delipidation.
5Avii	The used detergents are incompatible with Native MS. This often results into use of excessive collision energy to ablate the micelle. Beside the presence of highly charged species, ion-mobility based unfolding analysis can reveal the denatured state of the protein. 31	Often certain membrane proteins, in spite of being stable in certain detergents, do not yield good spectra under the nMS conditions. This is largely due to the stronger affinity of the specific protein for the detergent and/or large micelle size.	In these cases charge reducing agents (such as imidazole) can be added to the nMS electrospray buffer 32 or a different MS ion polarity can be used in an effort to stabilise the membrane protein complex.26 If this is not successful an additional screening process should be performed in an appropriate detergent for native MS, following our previous protocol. 20
5Bii	The HE platform is inefficient in removing the detergent micelle	It is feasible that the additional high energy available is still not sufficient to ablate the detergent micelle.	In our hands, using the high-energy activation method, we have been successful in liberating membrane protein-lipid complexes from various classes of commonly used detergents. However it is possible that some detergents may not be able to be removed even at very high activation energies. In such cases alternative detergents should be trialled.
5Biv	Lipid bound peak of interest could not be isolated for fragmentation	Due to presence of multiple peaks within close proximity at the m/z scale exclusive isolation of a single peak of interest may become difficult.	Check if the isolated spectrum contains only the lipid bound species, if more species are present reduce the quadrupole isolation window. A balance must be sought while selecting the window range. A large isolation window increases the intensity of the isolated peak but at the same time may include the non-lipid bound state or higher order lipid bound states.
5Bvi	Absence of signal at the lower m/z	Since the generated fragment ions of lipids or lower order oligomers are of lower m/z values than the parent ions, the instrumental condition set for the isolation of the parent ion may not optimum.	a. The trap pressure may need adjustment to ensure optimal transmission and detection of lower mass species. b. When increasing the trap energy to dissociate isolated protein-lipid complexes care must be taken to avoid increasing the energy so as to induce further dissociation and/or fragmentation of the product ions. This can lead to complex spectra that are difficult to interpret
5Bvi	The parent ion is recalcitrant to fragmentation	Due to greater Coulombic repulsion and the availability of larger numbers of mobile protons, higher charge states typically yield richer fragmentation spectra following CID. Nevertheless, a balance must be sought while choosing between the highest charge state and the parent ion intensity.	We suggest that experiments first be performed on the charge state with maximum intensity and if the resulting fragmentation pattern is not satisfactory, perform the isolation and fragmentation experiments on progressively higher charge states.