Depletion of internal peptides by site-selective blocking, phosphate labeling, and TiO2 adsorption for in-depth analysis of C-terminome.

The analysis of protein C-termini is of great importance, because it not only provides valuable information about protein function, but also facilitates the elucidation of proteolytic processing. However, even with the recent methods for the global profiling of protein C-termini, the identification of C-termini is still far behind that of N-termini due to the lack of basic residue and low reactive carboxyl group. Therefore, an unbiased and complementary method for C-termini profiling is imperative. In this work, we developed a negative enrichment strategy to achieve the in-depth analysis of C-terminome. Proteins were firstly amidated to block carboxyl groups, followed by lysyl endoproteinase (LysC) digestion to generate C-terminal peptides with α-amines and internal peptides bearing both α- and ε-amines. After the α-amines were blocked by site-selective dimethylation or succinylation, the remaining ε-amines on internal peptides were labeled with phosphate groups. Finally, internal peptides were depleted by TiO2, leaving exclusively the fraction of C-terminal peptides for LC-MS/MS analysis. With Escherichia coli (E. coli) digests as the sample, the efficiency of amidation, dimethylation/succinylation, phosphate labeling and TiO2 depletion was proved high. With the combination of dimethyl and succinic blocking strategy, our method enabled the identification of 477 unique C-terminal peptides in E. coli. In comparison with the C-terminal amine-based isotope labeling of substrates (C-TAILS) method, 83 C-termini were identified by both methods, whereas 369 C-termini were unique to C-TAILS and 394 to our dataset. The method proposed is therefore efficient and possibly promotes the comprehensive profiling of C-termini. Graphical Abstract Negative isolation of C-terminal peptides with combination of site-selective blocking, phosphate labeling, and TiO2 adsorption.

Age and composition of young basalts on the Moon, measured from samples returned by Chang'e-5.

Orbital data indicate that the youngest volcanic units on the Moon are basalt lavas in Oceanus Procellarum, a region with high levels of the heat-producing elements potassium, thorium, and uranium. The Chang'e-5 mission collected samples of these young lunar basalts and returned them to Earth for laboratory analysis. We measure an age of 1963 ± 57 million years for these lavas and determine their chemical and mineralogical compositions. This age constrains the lunar impact chronology of the inner Solar System and the thermal evolution of the Moon. There is no evidence for high concentrations of heat-producing elements in the deep mantle of the Moon that generated these lavas, so alternate explanations are required for the longevity of lunar magmatism.