New England Biolabs, Ipswich, MA, 01938, USA.
New England Biolabs, Ipswich, MA, 01938, USA; MCBB Graduate Program, Graduate School of Arts and Sciences, Boston University, Boston, MA, 02215, USA.
Anal Biochem. 2021 Jan 1;612:113761. doi: 10.1016/j.ab.2020.113761. Epub 2020 Jun 2.
Tubulin polyglutamylation is a polymeric modification that extends from the carboxyl-terminus of tubulins. Molecular description of amino acids and their branching polyglutamyls is a hallmark of tubulin in microtubules. There are different chemical approaches for detecting these polymeric structures, mostly reported prior to development of nESI peptide analysis. Here we demonstrate a novel and simple approach to detect shared regions of amino acid ions from tubulin polyglutamylated peptides in nanoLC-MS/MS. This involves two parallel in gel digestions with trypsin and subtilisin followed by mapping of di- and triglutamyl modifications of α- and β-tubulins using a routine proteomics assay. We present three levels of information: i) identification of proteomics MS/MS data, ii) description of internal fragment ion series common across digests, and iii) extracted ion chromatograms mapped relative to retention time standards for confirmation of relative hydrophobicity values. Our nanoLC assay positive ion ESI detects up to 3 conjugated glutamates in tubulins. We implemented an analytical column only bottom up approach that characterizes molecular features of polyglutamylated tubulins.
微管蛋白多聚谷氨酸化是一种从微管蛋白羧基末端延伸的聚合修饰。氨基酸及其分支多聚谷氨酸的分子描述是微管蛋白在微管中的特征。有不同的化学方法来检测这些聚合结构，其中大多数方法是在 nESI 肽分析之前报道的。在这里，我们展示了一种新颖而简单的方法，用于在纳升液相色谱-串联质谱 (nanoLC-MS/MS) 中检测微管蛋白多聚谷氨酸化肽中的氨基酸离子的共享区域。这涉及两种平行的胶内消化，分别用胰蛋白酶和枯草杆菌蛋白酶进行，然后使用常规蛋白质组学测定法对 α-和 β-微管蛋白的二聚体和三聚体修饰进行映射。我们提供了三个级别的信息：i）鉴定蛋白质组学 MS/MS 数据，ii）描述消化过程中常见的内部片段离子系列，以及 iii）提取离子色谱图相对于保留时间标准进行相对疏水性值的确认。我们的 nanoLC 测定正离子 ESI 检测到高达 3 个连接的谷氨酸在微管蛋白中。我们实施了仅从头开始的分析柱方法，该方法表征了多聚谷氨酸化微管蛋白的分子特征。
