Impact of Charge Reduction on Sequence Coverage of Proteins by 193 nm Ultraviolet Photodissociation

An ongoing goal of top-down mass spectrometry is to increase the performance for larger proteins. Using higher energy activation methods, like 193 nm ultraviolet photodissociation (UVPD), offers the potential to cause more extensive fragmentation of large proteins and thereby yield greater sequence coverage. Obtaining high sequence coverage requires confident identification and assignment of fragment ions, and this process is hampered by spectral congestion and low signal-to-noise ratio (S/N) of the fragment ions. Here we explore the use of charge reduction methods to produce lower charge states of large proteins to increase ion accumulation and generate lower charge states of fragment ions, ones that are naturally dispersed in the <i>m</i>/<i>z</i> domain to alleviate spectral congestion. UVPD of low charge states of enolase (47 kDa) and PRN-1 (63 kDa) resulted in sequence coverages as high as 47% (24+ charge state of enolase) and 23% (32+ of PRN-1) in comparison to 17% (55+ charge state of enolase) and 9% (55+ charge state of PRN-1) obtained for standard high charge states. Proton transfer charge reduction (PTCR) reactions were performed to further disperse fragment ions in the <i>m</i>/<i>z</i> domain and enhance identification. When employing PTCR after UVPD, sequence coverage was maximized for the highest charge states for enolase (55+, 67%) and PRN-1 (55+, 34%), confirming that charge reduction of fragment ions had a more notable impact on outcomes than charge reduction of precursor ions. Sequence coverages were increased further by combining results from electron transfer higher energy collision dissociation (EThcD) and UVPD (85% coverage for enolase and 52% coverage for PRN-1), bolstering the use of complementary MS/MS methods to yield greater dividends for top-down analysis of larger proteins.