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.