A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Mutations in Dynamic Structural Elements Alter the Kinetics and Fidelity of the Multifunctional Class II Lanthipeptide Synthetase, HalM2

“…The samples were thawed and subjected to online pepsin digestion (at 15 °C), followed by desalting and C 18 reverse phase chromatography at 0.4 °C as described in the Supporting Information to minimize deuterium back exchange. MS data were collected on a Synapt G2-Si instrument (Waters) operated in ion mobility mode as described previously to increase the peak capacity of the assay. , The uptake of deuterium by HalM2-derived peptides was then determined using DynamX 3.0 software (Waters) by measuring the shift in the isotope distribution of individual peptides relative to an undeuterated control. A detailed description of the HDX-MS sample preparation, instrumental settings, and data analysis procedures is provided in the Supporting Information.…”

Developments in Hydrogen/Deuterium Exchange Mass Spectrometry

“…A number of specific, highly detailed protocols with step-by-step details of how and why one does various things in HDX MS workflows were published during the review period, including the studies in Methods in Enzymology that were described in a previous section of this review. Some protocol highlights include a JoVE article with experimental details of how one lab performs its HDX MS, a methods/protocols for performing HDX MS labeling of oxygen sensitive proteins, a detailed protocol and HDX MS characterization pipeline with examples, a study describing techniques including both HDX MS and cross-linking MS, and a detailed description of recommended practices and theoretical aspects of studying protein complexes by HDX MS …”

“…A number of specific, highly detailed protocols with step-by-step details of how and why one does various things HDX MS workflows were published during the review period, including the studies in Methods in Enzymology that were described in a previous section of this review. Some protocol highlights include a JoVE article with experimental details of how one lab performs its HDX MS, 134 a methods/protocols for performing HDX MS labeling of oxygen sensitive proteins, 135 a detailed protocol and HDX MS characterization pipeline with examples, 136 a study describing techniques including both HDX MS and cross-linking MS, 137 and a detailed description of recommended practices and theoretical aspects of studying protein complexes by HDX MS. 138 There was a study 139 reporting the results of comparing HDX MS results across multiple laboratories given the same starting material, a well understood and standardized antibody fragment. While the starting material was well controlled, there was less control and standardization of the analysis parameters, instrumentation, and labeling methodology.…”

Biochemical and biophysical investigation of the HalM2 lanthipeptide synthetase using mass spectrometry

“…We developed an HDX-MS workflow for HalM2 that reproducibly generates over 200 HalM2derived peptides, provides nearly 90% sequence coverage, and allows measurement of deuterium uptake differences as small as 0.4 Da at the 99% confidence interval (Figure 8A). [40][41][42] The most dynamic regions of the free enzyme (e.g. highest deuterium uptake) include the Nterminal capping helices, the interface between the kinase activation domain and the activation loop, the interface between the cyclase domain and the dehydrase C-lobe, and a large loop spanning HalM2 residues P349-P405 (Figure 8B).…”

“…In Section 3, we briefly introduce the haloduracin  synthetase (HalM2) and its substrate peptide (HalA2) from Bacillus halodurans as a model system for elucidating structure-function relationships in class II lanthipeptide synthetases. Sections 4-6 highlight our systematic and detailed investigations into the HalM2:HalA2 system, 38,[40][41][42][43] and illustrate the many powerful and unique advantages of high-resolution mass spectrometry (MS) for characterizing the mechanisms of lanthipeptide biosynthesis. Namely, MS-based methods can assess every level of protein/peptide structure -all of which are relevant to understanding the mechanisms of lanthipeptide biosynthesis.…”