Differential isotope dansylation labeling combined with liquid chromatography mass spectrometry for quantification of intact and N-terminal truncated proteins

• LC–MS was developed for quantifying protein mixtures containing both intact and N-terminal truncated proteins.
• 12C2-Dansylation of the N-terminal amino acid of proteins was done first, followed by microwave-assisted acid hydrolysis.
• The released 12C2-dansyl labeled N-terminal amino acid was quantified using 13C2-dansyl labeled amino acid standards.
• The method provided accurate and precise results for quantifying intact and N-terminal truncated proteins within 8 h.

The N-terminal amino acids of proteins are important structure units for maintaining the biological function, localization, and interaction networks of proteins. Under different biological conditions, one or several N-terminal amino acids could be cleaved from an intact protein due to processes, such as proteolysis, resulting in the change of protein properties. Thus, the ability to quantify the N-terminal truncated forms of proteins is of great importance, particularly in the area of development and production of protein-based drugs where the relative quantity of the intact protein and its truncated form needs to be monitored. In this work, we describe a rapid method for absolute quantification of protein mixtures containing intact and N-terminal truncated proteins. This method is based on dansylation labeling of the N-terminal amino acids of proteins, followed by microwave-assisted acid hydrolysis of the proteins into amino acids. It is shown that dansyl labeled amino acids are stable in acidic conditions and can be quantified by liquid chromatography mass spectrometry (LC–MS) with the use of isotope analog standards.

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