Application of chemical selective cleavage methods to analyze post-translational modification in proteins

Abstract

Three chemical specific cleavage reactions, one for the carboxyl side of aspartyl peptide bonds, one for the carboxyl side of asparaginyl peptide bonds and another for the amino side of seryl/threonyl peptide bonds have been recently established. Additionally, these reactions simultaneously react on several post-translationally modified groups in peptides or proteins. The modified groups cover the external modifications N-formyl, N-acetyl, N-pyroglutamyi residues and C-terminal-alpha amide, as well as the internal modifications such as O-acetyl serine, phosphorylated serine/tyrosine, sulfonylated tyrosine, glycosylated serine/threonine and glycosylated asparagine. These three cleavage reactions relate to key amino acids for modifications, deamidation for asparagine, phosphorylation and acetylation for serine, and glycosylation for asparagine, serine and threonine. The chemical reactions on these modifications change the peptide mapping pattern, and information from these reactions may contribute characterization and location of post-translational modified groups in the protein.

The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites

“…Also, fragmentation spectra of peptides, where the modified site is located in the middle of the sequence, are more instructive. On the other hand, enzyme cleavage sites too close to the phosphorylated residues can reduce the cleavage efficiency [47,60]; therefore, a choice of different enzymes or chemical cleavage reactions might be considered [115]. Thirdly, dephosphorylation using alkaline phosphatase, both of the protein and its peptides, is an analytic tool for phosphoryla- The method has been miniaturised using disposable MALDI target plates constructed from either polymethylmethacrylate or polycarbonate in a nanovial array format [116].…”

Disulfide proteome in the analysis of protein function and structure

“…The application of established 2-DE methods of proteomics gives important additional data [1], but neglects information of great value in making functional assignments [4]. Parallel approaches may provide insight into structure and function that complement the electrophoresis data [5]. These include protein interactions, classification and post-translational modification of proteins.…”

Site-Specific Quantitation of Protein Nitration Using Liquid Chromatography/Tandem Mass Spectrometry

“…Therefore, an important emphasis of mass spectrometric studies of posttranslational modifications has been the development of methods that facilitate the detection of the modified peptides. Techniques such as affinity isolation chemistry [1][2][3] and selective detection based on characteristic fragmentation reactions [4][5][6] have been described as aids in this process. Once detected, these modified peptides are characterized by collision-induced dissociation (CID) in standard tandem mass spectrometry sequencing experiments.…”