Single nucleotide polymorphisms (SNPs) can result in protein sequence polymorphisms (PSPs) when codon translations are altered. Both top-down and bottom-up proteomics strategies can identify PSPs, but only if databases and software are used with this in mind. A 14319 Da protein from human saliva was characterized using the top-down approach on a hybrid linear ion-trap Fourier-transform ion cyclotron resonance mass spectrometer equipped for both collisionally-activated (CAD) and electron-capture (ECD) dissociation. Sequence tags identified the protein as Cystatin SN, and defined the N-terminal signal peptide cleavage site, as well as two disulfide bonds, in agreement with previous studies. The mass of the intact protein (< 5 ppm error) deviated from that calculated from the published gene sequence by 16.031 Da, and, based on CAD and ECD fragment ion assignments, it was concluded that the isoform of the protein analyzed carried a PSP at residue 11 such that the Pro translated from the genome was in fact Leu/Ile. An independently determined SNP (rs2070856) subsequently confirmed the genetic basis of the mass spectral interpretation and defined the residue as Leu. In another example, the PRP3 protein with mass ∼10,999 Da was found to be an isomeric/isobaric mixture of the reported sequence with PSPs D4N or D50N (rs1049112). Both CAD and ECD datasets support two phosphorylation sites at residues Ser8 and Ser22, rather than Ser17. In the context of discovery proteomics, previously undefined PSPs and PTMs will only be detected if the logic of data processing strategies considers their presence in an unbiased fashion.

中文翻译：

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使用自上而下的傅里叶变换离子回旋共振质谱法研究人类唾液蛋白质的蛋白质序列多态性和翻译后修饰


当密码子翻译改变时，单核苷酸多态性 (SNP) 会导致蛋白质序列多态性 (PSP)。自上而下和自下而上的蛋白质组学策略都可以识别 PSP，但前提是在使用数据库和软件时考虑到这一点。在配备碰撞激活 (CAD) 和电子捕获 (ECD) 解离的混合线性离子阱傅里叶变换离子回旋共振质谱仪上，采用自上而下的方法对人类唾液中的 14319 Da 蛋白质进行了表征。序列标签将该蛋白鉴定为半胱氨酸蛋白酶抑制剂 SN，并定义了 N 末端信号肽切割位点以及两个二硫键，与之前的研究一致。完整蛋白质的质量（< 5 ppm 误差）与根据已发表的基因序列计算出的质量偏差 16.031 Da，并且根据 CAD 和 ECD 片段离子分配，得出结论，所分析的蛋白质同种型在残基 11 使得从基因组翻译的 Pro 实际上是 Leu/Ile。随后独立确定的 SNP (rs2070856) 证实了质谱解释的遗传基础，并将该残基定义为 Leu。在另一个例子中，质量约为 10,999 Da 的 PRP3 蛋白被发现是所报告序列与 PSP D4N 或 D50N (rs1049112) 的同分异构/同量异位混合物。 CAD 和 ECD 数据集都支持残基 Ser8 和 Ser22（而不是 Ser17）处的两个磷酸化位点。在发现蛋白质组学的背景下，只有当数据处理策略的逻辑以公正的方式考虑它们的存在时，以前未定义的 PSP 和 PTM 才会被检测到。