Guanidination of the Bowman-Birk soybean inhibitor: Evidence for the tryptic hydrolysis of peptide bonds involving homoarginine

Seidl, Dinah S.; Liener, Irvin E.

doi:10.1016/0006-291x(71)90018-0

Cited by 17 publications

(5 citation statements)

References 13 publications

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“…It is reported that trypsin could cleave guanidinated peptides, − indicating that guanidination labeling might be promising to solve the above-mentioned missing cleavage problem in protein N-terminome analysis. Furthermore, guanidination could increase the ionization efficiency of peptides, which is beneficial to improve the MS-based peptide identification capacity. − By the present labeling method, only lysine ε-amines rather than all free amines could be blocked before removal by amine reactive scavenger materials, resulting in a loss of plenty of protein N-terminal peptides.…”

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confidence: 99%

Comprehensive Analysis of Protein N-Terminome by Guanidination of Terminal Amines

et al. 2019

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Protein N-termini and their modifications not only represent different protein isoforms but also relate to the functional annotation and proteolytic activities. Currently, negative selection methods, such as terminal amine isotopic labeling of substrates (TAILS), are the most popular strategy to analyze the protein N-terminome, in which dimethylation or acetylation modification is commonly used to block the free amines of proteome samples. However, after tryptic digestion, the generated long peptides, caused by the missing cleavage of blocked lysine, could hardly be identified by MS, which hindered the deep-coverage analysis of N-terminome. Herein, to solve this problem, we developed an approach, named terminal amine guanidination of substrates (TAGS). 1H-Pyrazole-1-carboxamidine was used to effectively guanidinate lysine ε-amines and N-terminal α-amines, followed by tryptic digestion to generate N-terminal peptides without free amines and internal peptides with free amines. Then, the internal peptides with free amines were removed by hyperbranched polyglycerol-aldehyde polymers (HPG-ALDs) to achieve the negative enrichment of N-terminome. By TAGS, not only the cleavage rate of blocked lysine could be improved, but also the ionization efficiency of tryptic peptides was increased. In comparison, 1814 and 1620 protein N-termini were, respectively, identified by TAGS and TAILS in Saccharomyces cerevisiae (S. cerevisiae). Among them, 1012 N-termini were uniquely identified in TAGS. Furthermore, by the combination of TAGS and the stable isotope labeling with amino acids in cell culture (SILAC)/label-free quantitative method, we not only identified the known N-terminal cleavage fragment of gasdermin D but also identified some new cleavage sites during Val-boroPro-induced pyroptosis. All these results demonstrated that our developed approach, TAGS, might be of great promise for the comprehensive analysis of N-terminome and beneficial for promoting the identification of protein isoforms and studying in-depth the proteolytic activity of proteins.

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confidence: 99%

Comprehensive Analysis of Protein N-Terminome by Guanidination of Terminal Amines

et al. 2019

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“…Trypsin cleavage of homoarginine‐modified proteins has been reported in the early literature . At this time, no proteomics study has taken this point into consideration when working on this modification.…”

Section: Resultsmentioning

confidence: 99%

Solid-phase N-terminal peptide enrichment study by optimizing trypsin proteolysis on homoarginine-modified proteins by mass spectrometry

Chowdhury

Munske

Yang

et al. 2014

Rapid Commun. Mass Spectrom.

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Rationale Proteolytic cleavages generate active precursor proteins by creating new N-termini in the proteins. A number of strategies recently published regarding the enrichment of original or newly formed N-terminal peptides using guanidination of lysine residues and amine reactive reagents. For effective enrichment of N-terminal peptides, the efficiency of trypsin proteolysis on homoarginine (guanidinated) modified proteins must be understood and simple and versatile solid-phase N-terminal capture strategies should be developed. Methods We present here a mass spectrometry-based study to evaluate and optimize the trypsin proteolysis on a guanidinated modified protein. Trypsin proteolysis was studied using different amount of trypsin to modified protein ratios. To capture the original N-termini, after guanidination of proteins, original N-termini were acetylated and the proteins were digested with trypsin. The newly formed N-terminal tryptic peptides were captured with a new amine reactive acid-cleavable solid-phase reagent. The original N-terminal peptides were then collected from the supernatant of the solution. Results We demonstrated a detailed study of the efficiency of enzyme trypsin on homoarginine modified proteins. We observed that the rate of hydrolysis of homoarginine residues compared to their lysine/arginine counter parts were slower but generally cleaved after an overnight digestion period depending on the protein to protease concentration ratios. Selectivity of the solid-phase N-terminal reagent was studied by enrichment of original N-terminal peptides from two standard proteins, ubiquitin and RNaseS. Conclusion We found enzyme trypsin is active in guanidinated form of protein depending on enzyme to protein concentrations, time and the proximity of arginine residues in the sequence. The novel solid-phase capture reagent also successfully enriched N-terminal peptides from the standard protein mixtures. We believe this trypsin proteolysis study on homoarginine modified proteins and our simple and versatile solid- phase capture strategy could be very useful for enrichment and sequence determination of proteins N-termini by mass spectrometry.

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“…An extensive search also revealed a few literature references which describe this tryptic hydrolysis of guanidinated proteins, although this topic is not their main focus. [40][41][42][43] The data in Table 1 can lead to several conclusions related to guanidination of proteins and tryptic hydrolysis of peptide bonds involving homoarginine generated from guanidination, although further investigation may be needed for the confirmation. (1) Most lysine residues were completely guanidinated and completely digested resulting in >95% yield of homoarginine peptides.…”

Section: Protein Guanidination and Tryptic Digestionmentioning

confidence: 99%

Guanidinated protein internal standard for immunoaffinity‐liquid chromatography/tandem mass spectrometry quantitation of protein therapeutics

Yang

Kernstock

Simmons

et al. 2014

Rapid Comm Mass Spectrometry

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Guanidination of the Bowman-Birk soybean inhibitor: Evidence for the tryptic hydrolysis of peptide bonds involving homoarginine

Cited by 17 publications

References 13 publications

Comprehensive Analysis of Protein N-Terminome by Guanidination of Terminal Amines

Comprehensive Analysis of Protein N-Terminome by Guanidination of Terminal Amines

Solid-phase N-terminal peptide enrichment study by optimizing trypsin proteolysis on homoarginine-modified proteins by mass spectrometry

Guanidinated protein internal standard for immunoaffinity‐liquid chromatography/tandem mass spectrometry quantitation of protein therapeutics

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