Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry.
Because of unparalleled sensitivity and tolerance to protein size, mass spectrometry (MS) has become a popular method for measuring the solution hydrogen (1H/2H) exchange (HX) of biologically relevant protein states. While incorporated deuterium can be localized to different regions by pepsin proteolysis of the labeled protein, the assignment of deuteriums to individual residues is typically not obtained, thereby limiting a detailed understanding of HX and the dynamics of protein structure. Here we use gas-phase fragmentation of peptic peptides by electron transfer dissociation (ETD) to measure the HX of individual amide linkages in the amyloidogenic protein beta2-microglobulin. A comparison of the deuterium levels of 60 individual backbone amides of beta2-microglobulin measured by HX-ETD-MS analysis to the corresponding values measured by NMR spectroscopy shows an excellent correlation. The deuterium labeling pattern of beta2-microglobulin is retained in the gaseous fragment ions by employing mild declustering conditions for electrospray ionization. A recently developed model peptide is used to arrive at such ion source declustering conditions that prevent the occurrence of intramolecular gas-phase hydrogen (1H/2H) migration (i.e., hydrogen scrambling). This article demonstrates that ETD can be implemented in a mass spectrometric method to monitor the conformational dynamics of proteins in solution at single-residue resolution.
Rand
Kasper D
KD
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. k.rand@neu.edu
Zehl
Martin
M
Jensen
Ole N
ON
Jørgensen
Thomas J D
TJ
D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement; D004579:Electron Transport; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012996:Solutions; D013379:Substrate Specificity; D001613:beta 2-Microglobulin / Q000737:chemistry*
D012996:Solutions; D001613:beta 2-Microglobulin