Mandell J G, Falick A M, Komives E A
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0601, USA.
Anal Chem. 1998 Oct 1;70(19):3987-95. doi: 10.1021/ac980553g.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was used to determine amide proton/deuteron (H/D) exchange rates. The method has broad application to the study of protein conformation and folding and to the study of protein-ligand interactions and requires no modifications of the instrument. Amide protons were allowed to exchange with deuterons in buffered D2O at room temperature, pD 7.25. Exchanged deuterons were "frozen" in the exchanged state by quenching at pH 2.5, 0 degree C and analyzed by MALDI-TOF MS. The matrix mixture consisted of 5 mg/mL alpha-cyano-4-hydroxycinnamic acid, acetonitrile, ethanol, and 0.1% TFA. The matrix was adjusted to pH 2.5, and the chilled MALDI target was rapidly dried. Deuteration of amide protons on cyclic AMP-dependent protein kinase was measured after short times of incubation in deuterium by pepsin protein digestion and MALDI-TOF MS analysis. The unseparated peptic digest was analyzed in a single spectrum of the mixture. From five spectra, H/D exchange rates were determined for some 40 peptides covering 65% of the protein sequence.
基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱法（MS）用于测定酰胺质子/氘核（H/D）交换率。该方法在蛋白质构象和折叠研究以及蛋白质-配体相互作用研究中具有广泛应用，且无需对仪器进行改装。在室温、pD 7.25的缓冲D2O中，使酰胺质子与氘核进行交换。通过在pH 2.5、0℃下淬灭，将交换后的氘核“冻结”在交换状态，并通过MALDI-TOF MS进行分析。基质混合物由5 mg/mLα-氰基-4-羟基肉桂酸、乙腈、乙醇和0.1% TFA组成。将基质调节至pH 2.5，并将冷却的MALDI靶快速干燥。通过胃蛋白酶消化蛋白质和MALDI-TOF MS分析，在氘中孵育短时间后，测定环磷酸腺苷依赖性蛋白激酶上酰胺质子的氘代情况。未分离的胃蛋白酶消化产物在混合物的单光谱中进行分析。从五个光谱中，测定了覆盖65%蛋白质序列的约40个肽段的H/D交换率。
