Limited Proteolysis with Targeted Proteomics Characterizes Protein Structure

Exploiting a feature of the protein characteristic under investigation, Feng et al. (2014) have developed a targeted proteomics workflow for large-scale investigation of structural changes.¹ The scientists used a limited proteolysis strategy for initial protein digestion, then completed their proteomic analysis with selected reaction monitoring (SRM)-based quantitation.

Protein function is a key regulator of protein activity; simple conformational changes determine access to substrates and receptors, thereby affecting cell processes. As Feng and colleagues point out, however, no efficient method currently exists for global screening on a large scale. Furthermore, analysis in complex biological matrices is also difficult. Although methods such as X-ray crystallography, nuclear magnetic resonance imaging and certain labeling techniques have been available for many years, they are not applicable to the large-scale, global proteomics research in vogue today.

Feng and co-authors devised a partial digestion strategy, using limited proteolysis where the enzymatic activity relied upon the structural conformation of the parent protein. The proteases explored included proteinase K, thermolysin and papain, used at very low concentrations and for extremely short durations (30 minutes, versus overnight). Once they quenched the reaction, the researchers performed subsequent digestion with trypsin and compared the peptide products with a full tryptic digest to evaluate structural and conformational influence on protease activity. Following this two-step, limited proteolysis method, they analyzed the digests with an LTQ Orbitrap Velos hybrid ion trap-Orbitrap mass spectrometer or a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (both Thermo Scientific). From this initial shotgun data, the researchers could then develop an SRM assay that quantified both peptides and conformational changes.

The team’s first experiment evaluated this limited proteolysis workflow, using the double digestion steps on human α-Syn, a primarily unfolded protein implicated in pathological changes associated with Parkinson’s disease, where the protein switches to β-sheet formation and polymerizes into fibrillar, amyloid aggregates. The researchers spiked monomeric, unfolded and induced fibrilized α-Syn into yeast total protein extracts as a complex matrix. Data from this work consistently mapped the proteolytic cleavage sites as predicted, showing that the technique was suitable for analyzing structural change.

Following these initial studies, Feng et al. looked to see if their method could detect subtle structural transformations, using myoglobin as an example. Change from holomyoglobin to apomyoglobin, as is seen with heme dissociation, is subtle, with altered conformation occurring in only one of its eight helices. Spiked into yeast proteome extracts, the experimental workflow gave results consistent with the known concentrations of the initial preparations.

Once fully validated, the scientists turned to large-scale analysis of global protein structural changes. They used the yeast species Saccharomyces cerevisiae and looked at protein structural alterations in response to nutritional change. Switching from glucose- to ethanol-based culture conditions causes protein conformational changes as cells adapt to different growth environments. Once analyzed, the researchers identified 21,899 peptides and 1,622 proteins containing 4,267 conformational sites, as mapped using the limited proteolysis strategy. From these data, they found that 586 of the peptides with identified conformation sites showed change in abundance with nutritional modulation, mapping to 283 proteins. Following further analysis with reference to Gene Ontology terms, the researchers found that most of the nutritionally modulated proteins were associated with catalytic activity and metabolic processes.

The team then explored structural change in key enzymes as a method of controlling activity. Looking at pyruvate kinase Cdc19, they found a response to fructose-1,6-bisphosphate (FBP) in the growth medium that explained the structural changes during the nutritional modulation experiments.

Using an SRM assay, the team quantified abundance of certain enzymes and also measured the structural changes. Interestingly, although structural change occurred in some enzymes, it was not a consistent finding across all metabolic pathways. Feng et al. suggest that this could indicate that structural change is not associated with regulation of enzyme activity in these experimental conditions.  

Conversely, SRM assay results also showed that structural changes occurred in some proteins even though there was no change in their overall abundance. When the researchers repeated the nutritional modulation experiments in mutant S. cerevisiae strains deficient in one of these proteins, Bmh1, they found poor growth in the ethanol-based culture conditions. They suggest that altered conformation of the protein is a key step for switching metabolism successfully.

Although Feng and colleagues acknowledge that some protein structural states may be resistant to proteolytic degradation, they postulate that their limited proteolysis–SRM workflow is a valuable tool for global assessment of the proteome. They suggest that for full evaluation, a proteome must be characterized in terms of its functionality as regulated by protein structure and conformation.

 

Reference

1. Feng, Y., et al. (2014, September) “Global analysis of protein structural changes in complex proteomes,” Nature Biotechnology, 32 (pp. 1036–44), doi: 10.1038/nbt.2999.

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