Hydrogen exchange monitored by MALDI‐TOF mass spectrometry for rapid characterization of the stability and conformation of proteins

Abstract

Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) has been used to monitor hydrogen exchange on entire proteins. Two alternative methods have been used to carry out the hydrogen exchange studies, exchanging deuteron (H to D experiments) or proton (D to H experiments). In the former case, the use of a deuterated matrix has made possible to overcome back-exchange problems and attain reproducible results. The methods presented have been used to determine the slow exchange core of the potato carboxypeptidase inhibitor in different folding states, and to differentially compare the activation domain of human procarboxypeptidase A2 versus three site-directed mutants of different conformational stability. In this work, we show that by using MALDI-TOF MS to monitor hydrogen exchange in entire proteins, it is possible to rapidly check the folding state of a protein and characterize mutational effects on protein conformation and stability, while requiring minimal amounts of sample.z 2000 Federation of European Biochemical Societies.

Role of Kinetic Intermediates in the Folding of Leech Carboxypeptidase Inhibitor

“…The conformational stability of the above-mentioned LCI folding intermediates was also investigated by D/H exchange experiments followed by MALDI-TOF/MS (42). The extent of hydrogen exchange was quite different for the native form and its folding intermediates.…”

Conformational Changes of Troponin C Within the Thin Filaments Detected by Neutron Scattering

“…Purified dTnC was then dialyzed against solution containing 20 mM Mops (pH 7.0), 90 mM KCl, 2 mM MgCl 2 , and 1 mM DTT. The degree of deuteration was determined from MALDI-TOF mass spectrometry 49 with an Applied Biosystems Voyager DE PRO.…”

Early Kinetics of Amyloid Fibril Formation Reveals Conformational Reorganisation of Initial Aggregates

“…In this work, we have chosen as target protein the activation domain of human procarboxypeptidase A2, ADA2h, which forms amyloid fibrils in vitro under certain experimental conditions, 6 and its folding pathway driving to the native state has been characterised extensively. [26][27][28][29][30][31][32][33] This 81-residue domain folds into two α-helices packing against a four-stranded β-sheet, as derived from the crystal structure of the complete proenzyme, 34 and from the NMR structure. 35 The comparison of both structures shows a slight destabilisation of β-strand 2 in the isolated domain due to the lack of specific contacts within the enzyme moiety.…”