How to format protein sequence?
If you have a sequence in a non-standard format you should:
- Save the sequence to a file as plain ASCII text, without any formatting whatsoever. ...
- If there is not an option to save your sequence in plain text format directly, there may well be a utility program to convert the file to plain text format. ...
- Use a text editor that is capable of writing files in plain text format in the future. ...
How to determine protein sequence?
• Protein is unknown or known, and comes from an organism with known DNA sequence –Purify protein (& separate chains) –Fragment chain(s) and sequence or measure mass –Use sequence database to reassemble sequence There are different approaches for using mass spectrometry to sequence a protein Bottom-Up Proteomics
What is the purpose of protein sequencing?
What are the Similarities Between DNA and Protein Sequence?
- Both DNA and protein sequences are large complex molecules.
- DNA contains protein-synthesizing genetic information.
- DNA sequences and protein sequences are building blocks of life.
What is protein sequencing and its methods?
SEQUENCING OF PROTEINS Protein sequencing is a technique to determine the amino acid sequence of a protein. It is a method to understand the structure and function of proteins in living organism. Amino acid sequence determines the eventual three dimensitional structure of the protein. I N T R O D U C T I O N 3 4.
Determining amino acid composition
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.
N-terminal amino acid analysis
Determining which amino acid forms the N -terminus of a peptide chain is useful for two reasons: to aid the ordering of individual peptide fragments' sequences into a whole chain, and because the first round of Edman degradation is often contaminated by impurities and therefore does not give an accurate determination of the N -terminal amino acid.
C-terminal amino acid analysis
The number of methods available for C-terminal amino acid analysis is much smaller than the number of available methods of N-terminal analysis.
Edman degradation
The Edman degradation is a very important reaction for protein sequencing, because it allows the ordered amino acid composition of a protein to be discovered. Automated Edman sequencers are now in widespread use, and are able to sequence peptides up to approximately 50 amino acids long.
Identification by mass spectrometry
Protein identification is the process of assigning a name to a protein of interest (POI), based on its amino-acid sequence. Typically, only part of the protein’s sequence needs to be determined experimentally in order to identify the protein with reference to databases of protein sequences deduced from the DNA sequences of their genes.
Bioinformatics tools
Bioinformatics tools exist to assist with interpretation of mass spectra (see De novo peptide sequencing ), to compare or analyze protein sequences (see Sequence analysis ), or search databases using peptide or protein sequences (see BLAST ).
Protein sequencing techniques
Nachimuthu Saraswathy, Ponnusamy Ramalingam, in Concepts and Techniques in Genomics and Proteomics, 2011
Mucosal Immunoglobulins
Immunochemical, protein sequencing, and molecular biology studies revealed the existence of IgA subclasses in several species, as mentioned above (for reviews, see Mestecky and Russell (1986), Mestecky et al. (1989), Kawamura et al. (1992), Ueda et al. (1988), Burnett et al. (1989), Vernersson et al. (2010) ).
Techniques for Protein Analysis
Gülay Büyükköroğlu, ... Candan Hızel, in Omics Technologies and Bio-Engineering, 2018
Modern Methods in Natural Products Chemistry
With respect to peptide/protein sequencing, ECD has some very attractive features, producing random cleavages along the peptide backbone (c - and z -type ions) and at the same time preserving labile PTMs such as phosphorylation.
Branched-Chain Amino Acids, Part B
Natalia Y. Kedishvili, ... Robert A. Harris, in Methods in Enzymology, 2000
Notation
The sequence of a protein is usually notated as a string of letters, according to the order of the amino acids from the amino-terminal to the carboxyl-terminal of the protein. Either a single or three-letter code may be used to represent each amino acid in the sequence.
Methods of Protein Sequencing
There are two main methods used to find the amino acid sequences of proteins. Mass spectrometry is the most common method in use today because of its ease of use. Edman degradation using a protein sequenator is the second method, which is most useful if the N-terminus of a protein needs to be characterized.
Amino Acid Composition and Analysis
The unordered composition of an amino acid is often useful information when attempting to determine the ordered sequence of the protein. This is because it can help identify errors and interpret ambiguous results. Additionally, the frequency of amino acids can also help to decide upon the protease that is more appropriate for the protein digestion.
What Are Proteins Made Of?
Proteins are polymers, meaning they are large molecules made up of many smaller molecules. The small molecules that make up proteins are called amino acids.
Different Types of Protein Structure
The structure of proteins is directly related to their function and may be primary, secondary, tertiary, or quaternary.
Classes of Protein Structure
The function of a protein depends heavily on its final structure. Tertiary and quaternary proteins are both functional proteins with a 3D structure. However, the type of structure can vary significantly between different proteins.
What is Protein Denaturation?
Proteins are only functional so long as they keep their 3D structure. If they are unfolded and lose their shape, they will no longer be functional.
Overview
Protein sequencing is the practical process of determining the amino acid sequence of all or part of a protein or peptide. This may serve to identify the protein or characterize its post-translational modifications. Typically, partial sequencing of a protein provides sufficient information (one or more sequence tags) to identify it with reference to databases of protein sequences derived fr…
Determining amino acid composition
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. no…
N-terminal amino acid analysis
Determining which amino acid forms the N-terminus of a peptide chain is useful for two reasons: to aid the ordering of individual peptide fragments' sequences into a whole chain, and because the first round of Edman degradation is often contaminated by impurities and therefore does not give an accurate determination of the N-terminal amino acid. A generalised method for N-terminal amino acid analysis follows:
C-terminal amino acid analysis
The number of methods available for C-terminal amino acid analysis is much smaller than the number of available methods of N-terminal analysis. The most common method is to add carboxypeptidases to a solution of the protein, take samples at regular intervals, and determine the terminal amino acid by analysing a plot of amino acid concentrations against time. This method will be very useful in the case of polypeptides and protein-blocked N termini. C-terminal …
Edman degradation
The Edman degradation is a very important reaction for protein sequencing, because it allows the ordered amino acid composition of a protein to be discovered. Automated Edman sequencers are now in widespread use, and are able to sequence peptides up to approximately 50 amino acids long. A reaction scheme for sequencing a protein by the Edman degradation follows; some of t…
Identification by mass spectrometry
Protein identification is the process of assigning a name to a protein of interest (POI), based on its amino-acid sequence. Typically, only part of the protein’s sequence needs to be determined experimentally in order to identify the protein with reference to databases of protein sequences deduced from the DNA sequences of their genes. Further protein characterization may include confirmation of the actual N- and C-termini of the POI, determination of sequence variants and i…
Predicting from DNA/RNA sequences
In biology, proteins are produced by translation of messenger RNA (mRNA) with the protein sequence deriving from the sequence of codons in the mRNA. The mRNA is itself formed by the transcription of genes and may be further modified. These processes are sufficiently understood to use computer algorithms to automate predictions of protein sequences from DNA sequences, such as from whole-genome DNA-sequencing projects, and have led to the generation of large d…
Bioinformatics tools
Bioinformatics tools exist to assist with interpretation of mass spectra (see De novo peptide sequencing), to compare or analyze protein sequences (see Sequence analysis), or search databases using peptide or protein sequences (see BLAST).
Notation
Methods of Protein Sequencing
- There are two main methods used to find the amino acid sequences of proteins. Mass spectrometry is the most common method in use today because of its ease of use. Edman degradation using a protein sequenator is the second method, which is most useful if the N-terminus of a protein needs to be characterized. It is helpful to know which amino acid is at the …
Amino Acid Composition and Analysis
- The unordered composition of an amino acid is often useful information when attempting to determine the ordered sequence of the protein. This is because it can help identify errors and interpret ambiguous results. Additionally, the frequency of amino acids can also help to decide upon the protease that is more appropriate for the protein digestion. There are two main steps t…
References
Further Reading