Optimizing identification and quantitation of 15N-labeled proteins in comparative proteomics

Comparative proteomics has emerged as a powerful approach to determine differences in protein abundance between biological samples. The introduction of stable-isotopes as internal standards especially paved the road for quantitative proteomics for comprehensive approaches to accurately determine protein dynamics. Metabolic labeling with (15)N isotopes is applied to an increasing number of organisms, including Drosophila, C. elegans, and rats. However, (15)N-enrichment is often suboptimal (<98%), which may hamper identification and quantitation of proteins. Here, we systematically investigated two independent (15)N-labeled data sets to explore the influence of heavy nitrogen enrichment on the number of identifications as well as on the error in protein quantitation. We show that specifically larger (15)N-labeled peptides are under-represented when compared to their (14)N counterparts and propose a correction method, which significantly increases the number of identifications. In addition, we developed a method that corrects for inaccurate peptide ratios introduced by incomplete (15)N enrichment. This results in improved accuracy and precision of protein quantitation. Altogether, this study provides insight into the process of protein identification and quantitation, and the methods described here can be used to improve both qualitative and quantitative data obtained by labeling with heavy nitrogen with enrichment less than 100%.