Laboratoire de Chimie Physique Moléculaire , École Polytechnique Fédérale de Lausanne , Station-6 , 1015 Lausanne , Switzerland.
Moscow Institute of Physics and Technology (State University) , 9 Institutskiy per. , Dolgoprudny , Moscow Region , 141701 , Russia.
Anal Chem. 2019 May 21;91(10):6709-6715. doi: 10.1021/acs.analchem.9b00770. Epub 2019 May 10.
Identification of isomeric amino acid residues in peptides and proteins is challenging but often highly desired in proteomics. One of the practically important cases that require isomeric assignments is that associated with single-nucleotide polymorphism substitutions of Met residues by Thr in cancer-related proteins. These genetically encoded substitutions can yet be confused with the chemical modifications, arising from protein alkylation by iodoacetamide, which is commonly used in the standard procedure of sample preparation for proteomic analysis. Similar to the genetically encoded mutations, the alkylation also induces a conversion of methionine residues, but to the iso-threonine form. Recognition of the mutations therefore requires isoform-sensitive detection techniques. Herein, we demonstrate an analytical method for reliable identification of isoforms of threonine residues in tryptic peptides. It is based on ultraviolet photodissociation mass spectrometry of cryogenically cooled ions and a machine-learning algorithm. The measured photodissociation mass spectra exhibit isoform-specific patterns, which are independent of the residues adjacent to threonine or iso-threonine in a peptide sequence. A comprehensive metric-based evaluation demonstrates that, being calibrated with a set of model peptides, the method allows for isomeric identification of threonine residues in peptides of arbitrary sequence.
鉴定肽和蛋白质中的异构氨基酸残基具有挑战性，但在蛋白质组学中通常是非常需要的。在需要进行异构分配的实际重要情况之一是与癌症相关蛋白质中 Met 残基被 Thr 的单核苷酸多态性取代有关。这些遗传编码的取代可能与化学修饰相混淆，这些修饰是由碘乙酰胺引起的蛋白质烷基化产生的，碘乙酰胺通常用于蛋白质组学分析样品制备的标准程序中。与遗传编码的突变类似，烷基化也会诱导蛋氨酸残基的转化，但转化为异苏氨酸形式。因此，识别这些突变需要对异构体敏感的检测技术。在此，我们展示了一种用于可靠鉴定胰蛋白酶肽中异苏氨酸残基的分析方法。它基于低温冷却离子的紫外光解离质谱和机器学习算法。所测量的光解离质谱表现出异构体特异性模式，这些模式与肽序列中邻近苏氨酸或异苏氨酸的残基无关。基于综合度量的评估表明，该方法在经过一组模型肽校准后，可以在任意序列的肽中进行异苏氨酸残基的鉴定。
