Validated determination of NRG1 Ig-like domain structure by mass spectrometry coupled with computational modeling.

High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure.

Rapid Quantification of Peptide Oxidation Isomers From Complex Mixtures.

Hydroxyl radical protein footprinting (HRPF) is a powerful technique for probing changes in protein topography, based on quantifying the amount of oxidation of different regions of a protein. While quantification of HRPF oxidation at the peptide level is relatively common and straightforward, quantification at the residue level is challenging because of the influence of oxidation on MS/MS fragmentation and the large number of complex and only partially chromatographically resolved isomeric peptide oxidation products. HRPF quantification of isomeric peptide oxidation products (where the peptide sequence is the same but isomeric oxidation products are formed at different sites) at the residue level by electron transfer dissociation tandem mass spectrometry (ETD MS/MS) has been demonstrated in both model peptides and HRPF products, but the method is hampered by the partial separation of oxidation isomers by reversed phase chromatography.

Variation in FPOP Measurements Is Primarily Caused by Poor Peptide Signal Intensity.

Fast photochemical oxidation of proteins (FPOP) may be used to characterize changes in protein structure by measuring differences in the apparent rate of peptide oxidation by hydroxyl radicals. The variability between replicates is high for some peptides and limits the statistical power of the technique, even using modern methods controlling variability in radical dose and quenching. Currently, the root cause of this variability has not been systematically explored, and it is unknown if the major source(s) of variability are structural heterogeneity in samples, remaining irreproducibility in FPOP oxidation, or errors in LC-MS quantification of oxidation.