Heavy Sugar and Heavy Water Create Tunable Intact Protein Mass Increases for Quantitative MS in any Feed and Organism

摘要

Stable isotope labeling techniques for quantitative top-down proteomics face unique challenges. These include unpredictable mass shifts following isotope labeling, which impedes analysis of unknown proteins and complex mixtures; and exponentially greater susceptibility to incomplete isotope incorporation, manifesting as broadening of labeled intact protein peaks. Like popular bottom-up isotope labeling techniques, most top-down labeling methods are restricted to defined media/feed as well as amino acid auxotrophic organisms. We present a labeling method optimized for top-down proteomics that overcomes these challenges. We demonstrated this method through the spiking of ¹³C-sugar or ²H-water into standard laboratory feedstocks, resulting in Tunable Intact Protein Mass Increases (TIPMI). After mixing of labeled and unlabeled samples, direct comparison of light and heavy peaks allowed for the relative quantitation of intact proteins in three popular model organisms, including prokaryotic and eukaryotic microorganisms, and an animal. This internal standard method proved to be more accurate than label-free quantitation in our hands. Advantages over top-down SILAC include working equally well in nutrient-rich media, conceivably expanding applicability to any organism and all classes of biomolecules; not requiring high resolving power MS for quantitation; and being relatively inexpensive.