Arginine-Selective Chemical Labeling Approach for Identification and Enrichment of Reactive Arginine Residues in Proteins

Modification of arginine residues
using dicarbonyl compounds is a common method to identify functional
or reactive arginine residues in proteins. Arginine undergoes several
kinds of posttranslational modifications in these functional residues.
Identifying these reactive residues confidently in a protein or large-scale
samples is a very challenging task. Several dicarbonyl compounds have
been utilized, and the most effective ones are phenylglyoxal and cyclohexanedione.
However, tracking these reactive
arginine residues in a protein or large-scale protein samples using
a chemical labeling approach is very challenging. Thus, the enrichment
of modified peptides will provide reduced sample complexity and confident
mass-spectrometric data analysis. To pinpoint arginine-labeled peptide
efficiently, we developed a novel arginine-selective enrichment reagent.
For the first time, we conjugated an azide tag in a widely used dicarbonyl
compound cyclohexanedione. This provided us the ability to enrich
modified peptides using a bio-orthogonal click chemistry and the biotin–avidin
affinity chromatography. We evaluated the reagent in several standard
peptides and proteins. Three standard peptides, bradykinin, substance
P, and neurotensin, were labeled with this cyclohexanedione-azide
reagent. Click labeling of modified peptides was tested by spiking
the peptides in a myoglobin protein digest. A protein, RNase A, was
also labeled with the reagent, and after click chemistry and biotin–avidin
affinity chromatography, we identified two selective arginine residues.
We believe this strategy will be an efficient way for identifying
functional and reactive arginine residues in a protein or protein
mixtures.

利用二羰基化合物（dicarbonyl compounds）对精氨酸残基（arginine residues）进行修饰，是鉴定蛋白质中功能性或反应性精氨酸残基的常用手段。这类功能性残基上的精氨酸可发生多种翻译后修饰（posttranslational modifications）。在蛋白质或大规模样本中精准鉴定这些反应性残基，是一项极具挑战性的工作。目前已开发出多种二羰基化合物，其中最为有效的为苯乙二醛（phenylglyoxal）与环己二酮（cyclohexanedione）。然而，通过化学标记策略追踪蛋白质或大规模蛋白质样本中的反应性精氨酸残基仍存在极大困难。因此，对修饰肽段进行富集可降低样本复杂度，助力获得可靠的质谱（mass-spectrometric）数据分析结果。为实现精氨酸标记肽段的高效富集，我们研发了一种新型精氨酸选择性富集试剂。我们首次将叠氮标签（azide tag）连接至广泛使用的二羰基化合物环己二酮上，由此可通过生物正交点击化学（bio-orthogonal click chemistry）与生物素-亲和素亲和层析（biotin–avidin affinity chromatography）技术实现修饰肽段的富集。我们在多种标准肽段与蛋白质中对该试剂进行了评估：选用缓激肽（bradykinin）、P物质（substance P）与神经降压素（neurotensin）三种标准肽段，使用该环己二酮-叠氮试剂进行标记；将标记后的肽段添加至肌红蛋白（myoglobin）酶解产物中，验证了修饰肽段的点击标记效果；同时用该试剂标记核糖核酸酶A（RNase A），经点击化学与生物素-亲和素亲和层析处理后，我们成功鉴定出两个选择性精氨酸残基。我们认为，该策略可高效实现蛋白质或蛋白质混合物中功能性与反应性精氨酸残基的鉴定。