Principle of Mass Spectrometer
A sizeable percentage of protein sequencing is accomplished using one of two major methods—mass spectrometry and the Edman degradation process. Mass spectrometry is one of the most direct methods and is gaining in popularity due to technological advances and improved computer technique, providing increased accuracy and sophistication. One weakness of this method is its increasing difficulty with increasing molecular size and reduced solubility. Molecules must be broken down and electrically charged, at which point they are forced into a magnetic field and deflected according to mass. One of the new methodologies is to break the protein down into smaller peptide units, and processing those.
The other main method used in protein sequencing is the Edman degradation reaction. This involves absorbing a protein and chemically modifying it, removing amino acids one at a time and making identification. One shortcoming of this procedure is it requires the amino group to be unmodified. Another difficulty is ascertaining the locations of disulfide bridges, if any. However, the efficiency of each step is approximately 98%, which allows around proteins of up to about 50 amino acids in length to be determined reliably.
It is also possible in some instances to deduce protein sequence from messenger RNA (mRNA) and DNA. How? Sequencing can be determined by mRNA through the process of gene transcription. This takes advantage of the following process: a gene transcription to an RNA molecule takes place, followed by the the removal of introns (segments of DNA that do not carry information relevant to protein synthesis) from between exons (which do carry relevant code) which are then spliced together by enzymes. The resultant species supplies the code whereby protein synthesis occurs from amino acid sequences.
Human Growth Hormone Amino Acid Sequence with Disulfide Bridge
Credit: National Institutes of Health (NIH)