Characterization of Unbranched Ubiquitin Tetramers by Combining Ultraviolet Photodissociation with Proton Transfer Charge Reduction Reactions.

ABSTRACT

Polyubiquitination is an important post-translational modification (PTM) that regulates various biological functions. The linkage sites and topologies of polyubiquitination chains are important factors in determining the fate of polyubiquitinated proteins. Characterization of polyubiquitin chains is the first step in understanding the biological functions of protein ubiquitination, but it is challenging owing to the repeating nature of the ubiquitin chains and the difficulty in deciphering linkage positions. Here, we combine ultraviolet photodissociation (UVPD) mass spectrometry and gas-phase proton transfer charge reduction (PTCR) to facilitate the assignment of product ions generated from Lys6-, Lys11-, Lys29-, Lys33-, Lys48-, and Lys63-linked ubiquitin tetramers. UVPD results in extensive fragmentation of intact proteins in a manner that allows the localization of PTMs. However, UVPD mass spectra of large proteins (>30 kDa) are often congested due to the overlapping isotopic distribution of highly charged fragment ions. UVPD + PTCR improved the identification of PTM-containing fragment ions, allowing the localization of linkage sites in all six tetramers analyzed. UVPD + PTCR also increased the sequence coverage obtained from the PTM-containing fragment ions in each of the four chains of each tetramer by 7 to 44% when compared to UVPD alone.