Table 2. Troubleshooting.

Step No.	Problem	Possible Reason	Possible Solutions
4	Decreased spectra quality at higher m/z.	For UHMR effective resolution at higher m/z is lower.	Change m/z window to encompass only high m/z values i.e. from 10,000 to 20,000 m/z. Set detector to high m/z. Increase HCD (UHMR) or backing (Synapt) pressure to stabilize high mass species.
1,4	Absence of signal.	Insufficient protein Vesicles do not exit capillary	Collect additional vesicles. Expand capillary orifice.
3,4	Broad peaks throughout.	Insufficient desalting, too much sonication, use of non-compatible compounds for native MS.	Perform additional washes of preparation. Reduce sonication amplitude. Avoid having DTT, TCEP, and other small molecules that might complicate the analysis.
4	Collision induced dissociation not possible.	Not enough energy used throughout. Total energy used is insufficient.	Raise voltage throughout where possible.
5	Soluble contaminants.	Vesicles not washed well enough. Some maybe intrinsic components of the system.	Additional wash stages can be presented into the protocol by repeating ultracentrifugation multiple times. Utilization of a concentrator tube with a molecular weight cutoff larger than the contaminant’s mass. Higher energy releases more membranes proteins, making them of higher representation in the spectra when compared to contaminants.