Fig. 2.

(a) The quantification of Ub peptides derived from tryptic digestion of human K48 multi-Ub chains. Eight peptides were detected including K48 linked peptides. K63 was detected in trace amounts. Mono and poly-Ub entities were detected at low amol levels. These quantifications were linear over 4 orders of magnitude. Quantification data are also shown using an SDS-gel simulation for better visualization. The lower band in the simulated SDS-gel represents mono-Ub and the smear on top represents unresolved poly-Ub chains; (b) same as (a), except that human K63 multi-Ub chains were analyzed. The SRM data were compared with linkage specific immunodetection of K63 linkages. K63 linkage was detectable at the 100 fmol level which is a 1000 fold lower than the limit of detection by immunoblotting. Ub was detected at the 100 amol level as shown in simulated SDS-gel images on top of SRM peptide measurements.