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

Abstract

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.

1,3‐Diketone‐Modified Nucleotides and DNA for Cross‐Linking with Arginine‐Containing Peptides and Proteins

“…To the best of our knowledge, there has been only one example of Arg targeting with DNA using 4,6‐dioxoheptylamido group attached to DNA postsynthetically to a 2′‐amino group of an 2′‐amino‐2′‐deoxyuridine which was chemically incorporated to the oligonucleotide (ON) through phosphoramidite chemistry [18] . In affinity labelling and bioconjugations, the most common reactive groups used to target Arg are glyoxals [19] and other 1,2‐dioxo derivatives [20] . Since these highly reactive species were difficult to attach to dNTPs for polymerase synthesis of reactive DNA, we focused on 1,3‐diketones and report here the synthesis of dNTP building blocks bearing 2,4‐pentandione (either linear linked to C1 or branched linked through C3), their use in the enzymatic synthesis of reactive DNA probes and comparison of reactivity with Arg‐containing peptides and proteins.…”

“…[18] In affinity labelling and bioconjugations, the most common reactive groups used to target Arg are glyoxals [19] and other 1,2-dioxo derivatives. [20] Since these highly reactive species were difficult to attach to dNTPs for polymerase synthesis of reactive DNA, we focused on 1,3-diketones and report here the synthesis of dNTP building blocks bearing 2,4-pentandione (either linear linked to C1 or branched linked through C3), their use in the enzymatic synthesis of reactive DNA probes and comparison of reactivity with Arg-containing peptides and proteins.…”

Affinity and chemical enrichment strategies for mapping low‐abundance protein modifications and protein‐interaction networks

“…For confident mass‐spectrometric identification and the reduction of sample complexity, an enrichment strategy aimed at the reactive arginine residue on peptides was reported by Wanigasekara et al. [52]. Arginine residues in three standard peptides neurotensin, bradykinin, and substance P were labeled with cyclohexanedione‐azide (CHD‐azide) reagent.…”

“…Selective identification of reactive arginines by labeling with a cyclohexadione‐azide, click chemistry with Alkyne‐PEG4‐Biotin and subsequent purification of labeled peptides by biotin‐avidin affinity chromatography [52]…”

1,3‐Diketone‐Modified Nucleotides and DNA for Cross‐Linking with Arginine‐Containing Peptides and Proteins

“… [18] In affinity labelling and bioconjugations, the most common reactive groups used to target Arg are glyoxals [19] and other 1,2‐dioxo derivatives. [20] Since these highly reactive species were difficult to attach to dNTPs for polymerase synthesis of reactive DNA, we focused on 1,3‐diketones and report here the synthesis of dNTP building blocks bearing 2,4‐pentandione (either linear linked to C1 or branched linked through C3), their use in the enzymatic synthesis of reactive DNA probes and comparison of reactivity with Arg‐containing peptides and proteins.…”