Neutron-Encoded Mass Signatures for Quantitative Top-Down Proteomics

Abstract

The ability to acquire highly accurate quantitative data is an increasingly important part of any proteomics experiment, whether shotgun or top-down approaches are used. We recently developed a quantitation strategy for peptides based on neutron encoding, or NeuCode SILAC, which uses closely spaced heavy isotope-labeled amino acids and high-resolution mass spectrometry to provide quantitative data. We reasoned that the strategy would also be applicable to intact proteins and could enable robust, multiplexed quantitation for top-down experiments. We used yeast lysate labeled with either 13C615N2-lysine or 2H8-lysine, isotopologues of lysine that are spaced 36 mDa apart. Proteins having such close spacing cannot be distinguished during a medium resolution scan, but upon acquiring a high-resolution scan, the two forms of the protein with each amino acid are resolved and the quantitative information revealed. An additional benefit NeuCode SILAC provides for top down is that the spacing of the isotope peaks indicates the number of lysines present in the protein, information that aids in identification. We used NeuCode SILAC to quantify several hundred isotope distributions, manually identify and quantify proteins from 1:1, 3:1, and 5:1 mixed ratios, and demonstrate MS2-based quantitation using ETD.

Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

“…Electron transfer dissociation (ETD) leverages electron-driven radical rearrangements to promote cleavage of N-C α bonds between amino acid residues, preserving labile post-translational modifications (PTMs) and providing extensive sequence-informative fragmentation of peptides and proteins [17–19]. Ideally suited for large, highly charged protein molecules, ETD has afforded important gains in top down proteomics, extending protein sequence coverage and enabling characterization of important PTMs and sequence variants [20–24]. …”

Proteomic techniques to probe the ubiquitin landscape

“…New methods for multiplexing samples like the NeuCode technique that is based on differences in the nuclear binding energy allow higher degrees of multiplexing while not increasing the complexity of the sample [48,135]. This will not only allow larger ubiquitination based data sets, but also allows for multiplexing for top-down applications as well [136].…”

Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice

“…NeuCode can also be used for other proteomics strategies including data-independent acquisition 19 , targeted proteomics 25 , and even top-down approaches 26,27 . In this protocol we will focus only on data-dependent shotgun proteomic applications and refer readers to the documents cited above for alternative proteomics applications.…”