Why Is Protein Sequencing Mainly Based on Acid Hydrolysis and Supplemented by Alkaline Hydrolysis
Protein sequencing predominantly relies on acid hydrolysis as the primary method, with alkaline hydrolysis serving as a supplementary approach for the following reasons:
Higher Efficiency of Acid Hydrolysis
Compared to alkaline hydrolysis, acid hydrolysis more efficiently cleaves peptide bonds, facilitating the breakdown of proteins into smaller peptides. This makes it the preferred method in protein sequencing. Under acidic conditions, peptide bonds are more susceptible to cleavage, generating peptide fragments that are easier to analyze.
Reduced Amino Acid Degradation in Acid Hydrolysis
Alkaline hydrolysis can cause irreversible chemical modifications to certain amino acids, potentially compromising sequencing accuracy. For instance, tryptophan is highly prone to degradation under alkaline conditions, which can lead to erroneous sequencing results. In contrast, acid hydrolysis induces significantly less degradation of amino acids, preserving their integrity for analysis.
Greater Compatibility of Acid Hydrolysis
Acid hydrolysis is more compatible with both laboratory protocols and subsequent mass spectrometry analysis. The peptide fragments produced through acid hydrolysis can be directly subjected to mass spectrometric analysis, whereas those generated by alkaline hydrolysis often require additional processing steps, such as neutralization and desalting, before analysis.
Despite acid hydrolysis being the predominant approach in protein sequencing, alkaline hydrolysis may be employed as a complementary technique in specific cases. For example, when acid hydrolysis negatively impacts certain amino acids (e.g., aspartic acid and glutamic acid, which are highly susceptible to hydrolysis in acidic environments) or when the isolation of specific peptide sequences or protein fragments is required, alkaline hydrolysis may provide a viable alternative.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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