自上而下的蛋白质组学表征和定量钙调蛋白精氨化

bioRxiv Pub Date : 2024-08-08 DOI:10.1101/2024.08.08.607245
Rick M. Searfoss, Xingyu Liu, Benjamin A. Garcia, Zongtao Lin
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引用次数: 0

摘要

精氨酰基转移酶 1(ATE1)的精氨酰化作用是在蛋白质 N 端或侧链的活性氨基酸(如 Glu 和 Asp)上添加精氨酸(Arg)。在小鼠模型中,ATE1 基因敲除(KO)后,全身去除精氨酸化会导致心脏缺陷,从而导致胚胎死亡。精氨酸化的生物学重要性促使人们采用自下而上的方法发现蛋白质上的精氨酸化位点。虽然自下而上的蛋白质组学在定位肽精氨化方面功能强大,但它缺乏在蛋白质水平上量化蛋白质形式的能力。在此,我们开发了一种自上而下的蛋白质组学工作流程,用于表征和量化钙调蛋白(CALR)的精氨酸化。为了生成完全精氨化的 CALR(R-CALR),我们在信号肽(AA1-17)后插入了一个 R 残基。在 ATE1 KO 细胞中过表达后,CALR 和 R-CALR 被亲和纯化,并在正向模式下通过 LCMS 进行分析。两种蛋白形式都显示出 27-68 的电荷状态,其中电荷 58 为最强烈的电荷状态。它们的电子激活解离(EAD)MS2 图谱显示,蛋白质的 N 端优先碎裂,产生了足够的 c 离子,有利于精氨化位点的精确定位。钙结合域(CBD)产生的特征离子最少,这可能是由于存在大量的大于 100 个 D 和 E 残基。紫外光解离(UVPD)与 EAD 和 ETD 相比,大大提高了 CBD 的序列覆盖率。这种方法可以鉴定和量化缺失、内源性(低)和高水平的 CALR 精氨化。据我们所知,我们的工作是首次将自上而下蛋白质组学应用于表征体外和体内翻译后精氨化。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation
Arginylation installed by arginyltransferase 1 (ATE1) features an addition of arginine (Arg) to the reactive amino acids (e.g., Glu and Asp) at the protein N-terminus or side chain. Systemic removal of arginylation after ATE1 knockout (KO) in mouse models resulted in heart defects leading to embryonic lethality. The biological importance of arginylation has motivated the discovery of arginylation sites on proteins using bottom-up approaches. While bottom-up proteomics is powerful in localizing peptide arginylation, it lacks the ability to quantify proteoforms at the protein level. Here we developed a top-down proteomics workflow for characterizing and quantifying calreticulin (CALR) arginylation. To generate fully arginylated CALR (R-CALR), we have inserted an R residue after the signaling peptide (AA1-17). Upon overexpression in ATE1 KO cells, CALR and R-CALR were purified by affinity purification and analyzed by LCMS in positive mode. Both proteoforms showed charge states ranging from 27-68 with charge 58 as the most intense charge state. Their MS2 spectra from electron-activated dissociation (EAD) showed preferential fragmentation at the protein N-terminals which yielded sufficient c ions facilitating precise localization of the arginylation sites. The calcium-binding domain (CBD) gave minimum characteristic ions possibly due to the abundant presence of >100 D and E residues. Ultraviolet photodissociation (UVPD) compared with EAD and ETD significantly improved the sequence coverage of CBD. This method can identify and quantify CALR arginylation at absence, endogenous (low), and high levels. To our knowledge, our work is the first application of top-down proteomics in characterizing post-translational arginylation in vitro and in vivo.
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