Sequencing Protein Amino Acid Sequences by Mass Spectrometry
In biological research, sequencing is commonly employed to ascertain the precise sequence of molecules such as DNA or RNA, while protein sequences are typically elucidated using mass spectrometry. In protein mass spectrometry, proteins are initially digested into smaller peptide fragments. The mass of these fragments is then measured to infer the original amino acid sequence of the protein.
Experimental Procedures
1. Initially, proteins are dissolved in a suitable buffer and digested into peptides through enzymatic digestion, chemical cleavage, or other methods.
2. Subsequently, these peptides are ionized—often via electrospray ionization or matrix-assisted laser desorption/ionization—and introduced into the mass spectrometer.
3. The mass spectrometer then measures the mass-to-charge ratio of the peptide ions, which aids in deducing their amino acid composition.
4. Finally, by comparing the data against existing databases, peptide sequences are identified, facilitating the deduction of the original protein's amino acid sequence.
Considerations
While mass spectrometry is a powerful and precise analytical tool, it is subject to certain limitations. For instance, some amino acids, such as leucine and isoleucine, possess identical masses and cannot be distinguished by mass spectrometry. Moreover, certain post-translational modifications of proteins may interfere with mass spectrometric analysis.
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