Top-Down Characterization of Protein Anions Using Ultraviolet Photodissociation Mass Spectrometry

Top-down proteomics is primarily performed using electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in the positive mode. Development of methods in the negative mode can potentially facilitate analysis of acidic proteome, but has been hampered by the low ionization efficiency and the lack of effective fragmentation methods for protein anions. Here, we investigate the performance of ultraviolet photodissociation (UVPD) for top-down analysis of protein anions. We employed organic bases as additives in solution to yield highly charged, nonadducted protein anions of high abundance. We compared UVPD with higher energy collisional dissociation (HCD) and activated electron photodetachment (a-EPD) for fragmentation of proteins ranging from 8.6 to 47 kDa. UVPD yielded abundant charge-reduced precursor radicals, in addition to numerous <i>a/x, b/y</i> and <i>c/z</i> fragment ions. UVPD offered 70-95% sequence coverage for proteins below 20 kDa regardless of the presence of disulfide bonds, and 30% coverage for the largest protein studied (47 kDa enolase), higher coverage than HCD and a-EPD. UVPD of deprotonated proteins exhibited several features similar to those of protonated proteins, such as minimal sensitivity to the charge state, production of abundant <i>a/x</i> fragment ions, and fairly uniform backbone cleavages adjacent to each residue (i.e., no prominent preferential cleavage sites).