Glocker M O, Borchers C, Fiedler W, Suckau D, Przybylski M
Fakultät für Chemie, Universität Konstanz, Germany.
Bioconjug Chem. 1994 Nov-Dec;5(6):583-90. doi: 10.1021/bc00030a014.
Amino-acetylation and -succinylation reactions in combination with mass spectrometric peptide mapping of tryptic peptide mixtures have been employed for surface topology-probing of lysine residues in bovine ribonuclease A, lysozyme, and horse heart myoglobin as model proteins of different surface structures. Direct molecular weight determinations identifying the precise number of acyl groups in partially modified proteins were obtained by electrospray and 252Cf-plasma desorption mass spectrometry. Electrospray mass spectra of multiply protonated molecular ions and deuterium exchange experiments provided a relative conformational characterization of protein derivatives and enabled the direct determinations of intact, partially acylated heme-myoglobin derivatives. Tryptic peptide mapping analysis, using plasma desorption and fast atom bombardment mass spectrometry, ascertained by mass spectrometric characterization of HPLC-separated modified peptides, yielded the exact identification of acylation sites. Relative reactivities of the amino acylation were derived from the peptide mapping data and from quantitative estimations of modified peptides upon acetylation/trideuteroacetylation and provided direct correlations with the relative surface accessibilities of lysine-epsilon-amino groups taken from X-ray crystallographic structure data of the proteins. The reactive lysine-41 residue in ribonuclease A which is part of the substrate binding site was directly identified from the mass spectrometric data. These results indicate tertiary structure-selective acylation combined with mass spectrometric peptide mapping as an efficient approach for the molecular characterization of surface topology and reactive fundamental lysine residues in proteins.
将氨基乙酰化和琥珀酰化反应与胰蛋白酶肽混合物的质谱肽图谱分析相结合，已用于对牛核糖核酸酶A、溶菌酶和马心肌红蛋白中的赖氨酸残基进行表面拓扑探测，这些蛋白作为具有不同表面结构的模型蛋白。通过电喷雾和252Cf等离子体解吸质谱法，直接测定了部分修饰蛋白质中酰基的确切数量，从而确定了分子量。多质子化分子离子的电喷雾质谱和氘交换实验提供了蛋白质衍生物的相对构象特征，并能够直接测定完整的、部分酰化的血红素-肌红蛋白衍生物。使用等离子体解吸和快原子轰击质谱法的胰蛋白酶肽图谱分析，通过对高效液相色谱分离的修饰肽进行质谱表征来确定，从而准确鉴定了酰化位点。氨基酰化的相对反应活性来自肽图谱数据以及乙酰化/三氘代乙酰化后修饰肽的定量估计，并与从蛋白质的X射线晶体结构数据中获取的赖氨酸-ε-氨基的相对表面可及性直接相关。从质谱数据中直接鉴定出核糖核酸酶A中作为底物结合位点一部分的活性赖氨酸-41残基。这些结果表明，三级结构选择性酰化与质谱肽图谱分析相结合，是一种用于蛋白质表面拓扑和活性碱性赖氨酸残基分子表征的有效方法。
