Heavy Sugar and Heavy Water Create Tunable Intact Protein Mass Increases for Quantitative Mass Spectrometry in Any Feed and Organism

Abstract

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.

Top-Down Proteomics: Ready for Prime Time?

“…174 Compared to SILAC, this method results in a more symmetric and predictable mass shift for heavy and light peak pairs, as the percentage of 13 C (or 2 H) incorporation in a protein deviates less than the percentage of lysine or arginine. 174 Although this labeling approach provides outstanding precision and accuracy, potential overlapping of the unlabeled proteoform peaks and the heavy peaks, and software requirement for picking labeled pairs still need to be addressed for large-scale quantitation. Therefore, the more accessible label-free techniques remain appealing in top-down quantitation proteomics.…”

Proteomes Are of Proteoforms: Embracing the Complexity

“…While the only truly absolute quantitative method, unfortunately, it is limited to cell cultures and not applicable to the vast bulk of proteomic studies that focus on tissues or biofluids [ 241 ]. Furthermore, SILAC introduces an additional shift in the isotopic envelope of the mass spectrum that may result in peak overlap which, coupled with the decreased likelihood of complete labelling as a function of increasing protein size, further complicates data analysis of larger proteins [ 242 ].…”

Metabolic labeling in middle-down proteomics allows for investigation of the dynamics of the histone code

“…Combining middle-down MS and metabolic labeling would be the first technique that allows discrimination of newly synthesized proteins or PTMs while preserving the information of coexisting PTM patterns. Interestingly, a few studies demonstrated that analysis of intact proteins, namely top-down MS, can be performed when such proteins are metabolically labeled [ 32 – 34 ]. However, histone analysis is a whole different issue, due to their very large degree of modified forms, many of them leading to the same intact protein mass.…”