Time-Dependent Changes in Side-Chain Solvent Accessibility during Cytochrome c Folding Probed by Pulsed Oxidative Labeling and Mass Spectrometry

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

“…Hydroxyl radical (·OH) is an interesting covalent probe due to its small size and high reactivity. Of the many methods for generating ·OH [13][14][15][16][17][18][19][20], the photolysis of H 2 O 2 by a pulsed UV laser [21][22][23] is particularly convenient. The microsecond ·OH lifetime implies that this photochemical technique is free of oxidation-induced artifacts if the measurements are conducted under single-exposure conditions [21,24,25].…”

Temporal Development of Protein Structure during S100A11 Folding and Dimerization Probed by Oxidative Labeling and Mass Spectrometry

“…54 Effective mixing and reproducible labeling were confirmed as described previously. 35,55 In addition, the mixer performance was verified optically in pH jump experiments on bromocresol purple. 21 We collected 250-μL portions of capillary outflow for each irradiated sample~1 s after mixing in microcentrifuge tubes that contained 13 μL of~150 mM phosphate buffer and 1.4 μM catalase at pH 7.…”

“…35 Both syringes were advanced simultaneously at 70 μL min − 1 using a syringe pump (Harvard Apparatus, Boston, MA). Syringe 1 contained acid-denatured S100A11 (50 μM, expressed on a monomer basis), 30 mM glutamine, 300 mM NaCl, and HCl at pH 2.…”

“…The digests were lyophilized and resuspended in 200 μL of water containing 10 μM bradykinin as an internal intensity standard. 35,55,58 Non-irradiated control samples were collected for every measurement by using the same solution conditions and mixing sequence, except that the laser was switched off.…”

“…31 Solvent accessibility patterns measured in this way are complementary to HDX data 34 and, advantageously, oxidative modifications can be introduced at neutral pH. The potential of ·OH labeling for timeresolved investigations is only beginning to emerge, [35][36][37][38][39] and never before has this approach been applied to rapid self-assembly processes of multi-subunit proteins.…”