Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry
Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry
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DOI：
10.1021/ac980553g
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发表时间：
1998-10-01
影响因子：
7.4
通讯作者：
Komives, EA
中科院分区：
文献类型：
--
作者：
Mandell, JG;Falick, AM;Komives, EA
采用基质辅助激光解吸电离飞行时间质谱法（MALDI-TOF MS）测定酰胺质子/氘（H/D）交换速率，该方法不需要对仪器进行任何改动，可广泛应用于蛋白质构象和折叠的研究以及蛋白质-配体相互作用的研究。使酰胺质子在室温下在缓冲的D2 O中与氘交换，pD 7.25。通过在pH 2.5、0 ℃下淬灭将交换的氘“冷冻”在交换状态，并通过MALDI-TOF MS分析。基质混合物由5 mg/mL α-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。通过胃蛋白酶蛋白消化和MALDI-TOF MS分析，在氘中短时间孵育后测量环AMP依赖性蛋白激酶上的酰胺质子的氘代。在混合物的单一光谱中分析未分离的消化物。从五个光谱，H/D交换率确定约40肽覆盖65%的蛋白质序列。
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 degrees 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.