Identification of ubiquitin sites on TDP-43 by mass spectrometry.

<p>(<b>A</b>) Trypsin digestion of ubiquitin conjugates generates a di-glycine tag (GG), with a monoisotopic mass of 114.0429 Da) on ubiquitin-modified lysine residues, producing unique MS/MS spectra that can be matched by SEQUEST. Searching against a concatenated target-decoy database followed by manual validation, three sites of lysine ubiquitination (K102, K114 and K181) were mapped to TDP-43 in the TDP-S6 insoluble proteome. A fourth site (K145) identified later is also shown. (<b>B</b>) Ubiquitination site context in the linear domain structure of TDP-S6 is shown. (<b>C</b>) Two views rotated about the y-axis of a three-dimensional model of RRM-1 docked to HIV TAR ssDNA (blue) modified from Kuo, et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038658#pone.0038658-Kuo1" target="_blank">[17]</a>. K102, K114, K145, and K181 side chains are labeled and explicitly shown (purple) in the context of the RRM-1 domain structure. The closest ε-amino group to the nucleic acid backbone (3.1 Å) was K181, which resides in the loop that follows the RRM1 C-terminus.