Editing Protein sequencing (section)

== Determining amino acid composition ==
[[File:Schermata 2022-06-24 alle 23.06.28.png|thumb|Protein sequence interpretation: a scheme new protein to be engineered in a yeast]]
It is often desirable to know the unordered amino acid composition of a protein prior to attempting to find the ordered sequence, as this knowledge can be used to facilitate the discovery of errors in the sequencing process or to distinguish between ambiguous results. Knowledge of the frequency of certain amino acids may also be used to choose which [[protease]] to use for digestion of the protein. The misincorporation of low levels of non-standard amino acids (e.g. norleucine) into proteins may also be determined.<ref>{{cite journal | vauthors = Bogosian G, Violand BN, Dorward-King EJ, Workman WE, Jung PE, Kane JF | title = Biosynthesis and incorporation into protein of norleucine by Escherichia coli | journal = The Journal of Biological Chemistry | volume = 264 | issue = 1 | pages = 531–9 | date = January 1989 | doi = 10.1016/S0021-9258(17)31291-7 | pmid = 2642478 | doi-access = free }}</ref> A generalized method often referred to as ''amino acid analysis''<ref name="AltermanHunziker2011">{{cite book|author1=Michail A. Alterman|author2=Peter Hunziker|title=Amino Acid Analysis: Methods and Protocols|url=https://books.google.com/books?id=CLdSXwAACAAJ|date=2 December 2011|publisher=Humana Press|isbn=978-1-61779-444-5}}</ref> for determining amino acid frequency is as follows:
# Hydrolyse a known quantity of protein into its constituent amino acids.
# Separate and quantify the amino acids in some way.

=== Hydrolysis ===
[[Hydrolysis]] is done by heating a sample of the protein in 6 M [[hydrochloric acid]] to 100–110&nbsp;°C for 24 hours or longer. Proteins with many bulky [[hydrophobic]] groups may require longer heating periods. However, these conditions are so vigorous that some amino acids ([[serine]], [[threonine]], [[tyrosine]], [[tryptophan]], [[glutamine]], and [[cysteine]]) are degraded. To circumvent this problem, Biochemistry Online suggests heating separate samples for different times, analysing each resulting solution, and extrapolating back to zero hydrolysis time. Rastall suggests a variety of reagents to prevent or reduce degradation, such as [[thiol]] [[reagent]]s or [[phenol]] to protect tryptophan and tyrosine from attack by chlorine, and pre-oxidising cysteine. He also suggests measuring the quantity of [[ammonia]] evolved to determine the extent of [[amide hydrolysis]].

=== Separation and quantitation ===
The amino acids can be separated by [[ion-exchange chromatography]] then derivatized to facilitate their detection. More commonly, the amino acids are derivatized then resolved by [[High-performance liquid chromatography#Reversed-phase chromatography .28RPC.29|reversed phase HPLC]].

An example of the ion-exchange chromatography is given by the NTRC using sulfonated polystyrene as a matrix, adding the amino acids in acid solution and passing a buffer of steadily increasing [[pH]] through the column. Amino acids are eluted when the pH reaches their respective [[isoelectric point]]s. Once the amino acids have been separated, their respective quantities are determined by adding a reagent that will form a coloured derivative. If the amounts of amino acids are in excess of 10 nmol, [[ninhydrin]] can be used for this; it gives a yellow colour when reacted with proline, and a vivid purple with other amino acids. The concentration of amino acid is proportional to the absorbance of the resulting solution. With very small quantities, down to 10 pmol, fluorescent derivatives can be formed using reagents such as [[phthaldehyde|ortho-phthaldehyde (OPA)]] or [[fluorescamine]].

Pre-column derivatization may use the Edman reagent to produce a derivative that is detected by UV light. Greater sensitivity is achieved using a reagent that generates a fluorescent derivative. The derivatized amino acids are subjected to reversed phase chromatography, typically using a C8 or C18 [[Column chromatography|silica column]] and an optimised [[Eluent|elution]] gradient. The eluting amino acids are detected using a UV or fluorescence detector and the peak areas compared with those for derivatised standards in order to quantify each amino acid in the sample.