GAS PHASE ANALYTICAL CHEMISTRY OF PEPTIDE IONS

低能碰撞活化分解/串联技术的应用 用质谱学(MS)对多肽进行表征是目前 受限于对多肽的气相化学的不完全理解 离子。我们最新的人胰蛋白酶多肽的串联MS分析 一架B-100和合成类似物一起揭示了意想不到的 质子化离子的碎裂行为。因此，半胱氨酸的氧化 到含有C-末端精氨酸残基的多肽中的半胱酸， 显著促进C末端(“Y”系列)碎片离子，产生清晰的 序列信息。这些数据与离子内的 氨基酸侧链之间的相互作用，促进了 [M+H]+物种中的电荷位置。我们还调查了其他 质子化和金属阳离子化的低能分解 多肽。质子化多肽的C-末端重排(丢失 C-末端氨基酸残基但保留羧基氧)具有 用[18O]标记和MS/MS/MS[18O]同位素交换研究 气相也得到了证实。C-末端的倾向 重排和[18O]交换是依赖于序列的。总而言之， 我们的数据表明了一个具有广泛分析意义的假设，即 质子化和阳离子金属的低能碎裂 电荷位置和构象的位置(部分由 质子化或其他阳离子)由气体中的多肽离子采用 相位。拟议的研究将通过以下方式评估这一假设 用快原子轰击分析选定的寡肽--和 电喷雾串联MS(包括MS/MS/MS技术 产物离子扫描和反应中间扫描)。我们会 研究不同的一系列多肽的碎片 尊重其组成的质子(或金属阳离子)亲和力 氨基酸和构象方面。选择性衍生化 将评估这些程序对气相质子和金属的影响 阳离子亲和力和寡肽的碎裂。我们的研究 将指导分析战略，以 修饰后的载脂蛋白B-100胰酶片段的特性 从氧化型人低密度脂蛋白(LDL)中提取。特别关注 将重视对氧化半胱氨酸和蛋氨酸的认识 残留物和与以下相关的修饰的表征 蛋白质与低密度脂蛋白氧化脂质成分的相互作用。因此， 与丙二醛结合的胰酶片段和 免疫亲和萃取法选择性分离4-羟基壬烯醛 对于串联MS的表征，基本的和 这里提出的分析研究解决了我们的基本前提 熟练使用串联质谱仪进行多肽结构表征 依赖于对多肽的气相化学的更好的理解 离子。

The application of low energy collisionally activated decomposition/tandem mass spectrometry (MS) to the characterization of peptides is currently limited by imperfect understanding of the gas-phase chemistry of peptide ions. Our recent tandem MS analyses of tryptic peptides derived from human a B-100, together with synthetic analogues, have revealed unexpected fragmentation behavior of the protonated ions. Thus, oxidation of cysteine to cysteic acid in peptides containing a C-terminal arginine residue, markedly promotes C-terminal ("Y" series) fragment ions, yielding clear sequence information. The data are consistent with an intra-ionic interaction between amino acid side-chains, promoting a multiplicity of sites of charge in the [M + H]+ species. We have also investigated other aspects of the low energy decomposition of protonated and metal-cationized peptides. A C-terminal rearrangement of protonated peptides (with loss of the C-terminal amino acid residue but retention of a carboxyl oxygen) has been studied using [18O]-labelling and MS/MS/MS. [18O] isotope exchange in the gas phase has also been demonstrated. The propensities to C-terminal rearrangement and [18O] exchange are sequence dependent. In combination, our data suggest a hypothesis of broad analytical significance, namely that low energy fragmentations of protonated and metal cationized by the site or sites of charge location and by the conformation (in part induced by protonation or other cationization) adopted by the peptide ion in the gas phase. The proposed research will evaluate this hypothesis through analyses of selected oligopeptides using fast atom bombardment- and electrospray-tandem MS (including MS/MS/MS techniques such as sequential product ion scanning and reaction intermediate scanning). We will investigate the fragmentations of series of peptides which differ with respect to the proton (or metal cation) affinities of their constituent amino acids and with respect to conformation. Selected derivatization procedures will be evaluated for their effect on gas phase proton and metal cation affinities and on the fragmentations of oligopeptides. Our studies of synthetic peptides will guide analytical strategies for the characterization of tryptic fragments of modified apoprotein B-100 derived from oxidized human low density lipoprotein (LDL). Particular attention will be paid to the recognition of oxidized cysteine and methionine residues and to the characterization of modifications associated with interaction of the protein with oxidized lipid constituents of LDL. Thus, tryptic fragments incorporating conjugation with malondialdehyde and 4-hydroxynonenal will be selectively isolated by immunoaffinity extraction for characterization by tandem MS. The combination of fundamental and analytical studies proposed here addresses our basic premise that proficiency in peptide structural characterization using tandem MS is dependent on improved understanding of the gas-phase chemistry of peptide ions.