The protein sequence is a practical process for determining the amino acid sequence of all or part of a protein or peptide. This may serve to identify proteins or characterize their post-transplant modification. Typically, partial protein sequencing provides sufficient information (one or more sequence tags) to identify it by reference to a protein sequence database derived from a conceptual translation of a gene.
The two main direct methods of protein sequencing are mass spectrometry and Edman's degradation using sequencers. The current mass spectrometry method is most widely used for sequencing and protein identification but Edman degradation remains a valuable tool for characterizing the N -usminus protein.
Video Protein sequencing
Determining amino acid composition
It is often desirable to know the irregular amino acid composition of a protein before attempting to find an ordered sequence, since 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 can also be used to select proteases used for protein digestion. Deviations of low non-standard amino acid levels (eg, norleucine) into proteins can also be determined. The common method often referred to as amino acid analysis to determine the frequency of amino acids is as follows:
- Hydrolyse a number of known proteins into their constituent amino acids.
- Separate and measure amino acids in some way.
Hydrolysis
Hydrolysis is carried out by heating the protein sample in 6 M hydrochloric acid up to 100-110 Â ° C for 24 hours or longer. Proteins with many large hydrophobic groups may require longer warming periods. However, this condition is so strong that some amino acids (serine, threonine, tyrosine, tryptophan, glutamine, and cysteine) are degraded. To solve this problem, Biochemistry Online suggests heating separate samples for different times, analyzing each solution produced, and extrapolating back to zero hydrolysis time. Rastall recommends various reagents to prevent or reduce degradation, such as thiol or phenol reagents to protect tryptophan and tyrosine from chlorine attacks, and pre-oxidation cysteine. He also suggested measuring the quantity of ammonia evolved to determine the degree of hydrolysis of the amide.
Separation and quantization
Amino acids can be separated by ion exchange chromatography and then derivatized to facilitate their detection. More generally, the derivatized amino acids are then solved with reversed phase HPLC.
Examples of ion exchange chromatography are given by NTRC using sulfonated polystyrene as a matrix, adding amino acids in acidic solutions and passing the increasing pH buffer through the column. Amino acids are eluted when the pH reaches each isoelectric point. After the amino acids have been separated, each quantity is determined by adding reagents that will form a colored derivative. If the amount of amino acids is more than 10 nmol, ninhydrin may be used for this; gives yellow color when reacting with proline, and purple lives with other amino acids. The amino acid concentration is proportional to the absorbance of the resulting solution. With very small amounts, up to 10 pmol, fluorescent derivatives can be formed using reagents such as ortho-phthaldehyde (OPA) or fluorescamine.
Pre-column derivatization can use the Edman reagent to produce derivatives detected by UV light. Greater sensitivity is achieved using reagents that produce fluorescent derivatives. The amino acid derivatisation is subjected to reversed phase chromatography, usually using C8 or C18 silica columns and elution gradients optimized. The amino acid eluting was detected using UV or fluorescence detector and peak areas compared to those for standard derivatization to measure each of the amino acids in the sample.
Maps Protein sequencing
N -terminal analysis of amino acids
Determining which amino acids make up the N -usine peptide chain is useful for two reasons: it helps to compile the order of individual peptide fragments into the entire chain, and because the first round of degradation of Edman is often contaminated by impurities and therefore not provides an accurate determination of N -inhibits of amino acids. Common methods for N - the following amino acid analysis terminals:
- Reacts peptides with reagents that will selectively label the terminal amino acids.
- Hydrolyse protein.
- Determine amino acids with chromatography and comparison with standards.
There are many different reagents that can be used to label terminal amino acids. They all react with the amine group and therefore will also bind the amine group on the amino acid side chain like lysine - for this reason it is necessary to be careful in interpreting the chromatogram to ensure that the right place is selected. Two of the more common reagents are Sanger Reagents (1-fluoro-2,4-dinitrobenzene) and derivative derivatives such as dansil chloride. Phenylisothiocyanate, the reagent for Edman degradation, can also be used. The same question applies here as in the determination of the amino acid composition, with the exception that no stain is required, since the reagents produce colored derivatives and only qualitative analysis is required. So the amino acids should not be eluted from the chromatographic column, just compared to the standard. Another consideration to consider is that, since each amine group will react with the labeling reagent, ion exchange chromatography is unusable, and thin layer chromatography or high pressure liquid chromatography should be used instead.
Analysis of C-terminal amino acids
The number of methods available for C-terminal amino acid analysis is much smaller than the number of methods available from N-terminal analysis. The most common method is to add carboxypeptidases to protein solution, take samples periodically, and determine terminal amino acids by analyzing plots of amino acid concentrations over time. This method will be very useful in case of polypeptide and protein-blocked N termini. C-terminal sequencing will be helpful in verifying the predominant protein structure predicted from DNA sequences and for detecting any post-transport process of gene products from known codon sequences.
Degradation of Edman
Edman degradation is a very important reaction for protein sequencing, because it allows the regular amino acid composition of the protein to be found. Edman's automatic sequences are now widely used, and can sequentially peptide up to about 50 amino acids. The reaction scheme for protein sequencing by Edman's degradation follows; some steps are outlined later.
- Destroy all disulfide bridges in proteins with reducers such as 2-mercaptoethanol. Protective groups such as iodoacetic acid may be necessary to prevent bonds from being re-established.
- Separate and clean individual chains of the protein complex, if there is more than one.
- Determine the amino acid composition of each chain.
- Determine the terminal amino acids of each chain.
- Separate each chain into fragments below 50 amino acids.
- Separate and clean the fragments.
- Specify the order of each fragment.
- Repeat with a different cleavage pattern.
- Create a sequence of whole proteins.
Digestion becomes a peptide fragment
Peptides longer than about 50-70 amino acids can not be sequenced reliably by Edman degradation. Therefore, long protein chains need to be broken down into small pieces that can then be sorted individually. Digestion is performed either by endopeptidases such as trypsin or pepsin or by chemical reagents such as cyanogen bromide. Different enzymes provide different cleavage patterns, and the overlap between the fragments can be used to build the overall sequence.
Reactions
The peptides to be sorted are adsorbed onto a solid surface. One of the common substrates is glass fibers coated with polybrene, cationic polymers. The Edman reagent, phenylisothiocyanate (PITC), is added to the adsorbed peptide, together with a somewhat basic buffer solution of 12% trimethylamine. It reacts with the amino group amino acid N-terminal.
Terminal amino acids can be selectively detached by addition of anhydrous acids. The derivative is then isomerised to provide substituted phenilthiohidantoin, which can be washed and identified by chromatography, and the cycle can be repeated. The efficiency of each step is about 98%, which allows about 50 amino acids to be reliably determined.
Sequence proteins
A protein sequenator is a machine that automatically degrades Edman. Samples of proteins or peptides immobilized in reaction vessels from protein sequencers and Edman degradation were performed. Each cycle releases and releases an amino acid from a protein or peptide N -minus and the derived amino acid derivative is then identified by HPLC. The sequencing process is performed repeatedly for the entire polypeptide until the entire measured sequence is set or for a predetermined number of cycles.
Identification with mass spectrometry
Identification of proteins is the process of naming for the desired protein (POI), based on the amino acid sequence. Usually, only part of the protein sequence needs to be determined experimentally to identify proteins with reference to the protein sequence data inferred from the DNA sequence of their genes. Further protein characterization may include confirmation of N- and C-termini from POI, determination of sequence variants and identification of existing post-translational modifications.
Proteolytics digest
A common scheme for protein identification is described.
- POIs are isolated, usually with SDS-PAGE or chromatography.
- The isolated POI can be chemically modified to stabilize the cysteine ​​residue (eg S-amidomethylation or S-carboxymethylation).
- POI is digested with specific proteases to produce peptides. Trypsin, which cuts selectively on the C-terminal side of Lysine or Arginine residues, is the most commonly used protease. The advantages include i) the residual frequency of Lys and Arg in proteins, ii) the high specificity of the enzyme, iii) the stability of the enzyme and iv) the suitability of tryptic peptides for mass spectrometry.
- Peptides can be removed to remove ionized contaminants and undergo MALDI-TOF mass spectrometry. Direct peptide mass measurements can provide enough information to identify proteins (see Peptide mass fingerprints) but peptide fragmentation in the mass spectrometer is more commonly used to obtain information about peptide sequences. Alternatively, the peptide can be removed and separated by reversed phase HPLC and inserted into a mass spectrometer via an ESI source. LC-ESI-MS can provide more information than MALDI-MS for protein identification but uses more instrument time.
- Depending on the type of mass spectrometer, peptide ion fragmentation can occur through various mechanisms such as collision Dissociation (CID) or Post-source decay (PSD). In each case, the peptide ion fragment pattern provides information about the sequence.
- The information including the measured mass of the suspected peptide ion and its fragment ions is then matched to the calculated mass value of the conceptual protein (in-silico) and protein sequence database fragmentation. A successful match will be found if the score exceeds the threshold based on the analysis parameters. Even if the actual protein is not represented in the database, fault tolerant matching allows for the identification of putative proteins based on similarities with homologous proteins. A variety of software packages are available to perform this analysis.
- Software packages usually generate reports that indicate the identity (access code) of each identified protein, matching scores, and provide a relative strength measure of matching in which some proteins are identified.
- Suitable peptide diagrams in identified protein sequences are often used to indicate sequence coverage (% of proteins detected as peptides). Where POI is considered to be significantly smaller than a suitable protein, the diagram may indicate whether POI is the N or C terminal fragment of the identified protein.
Sequence n novo
The peptide fragmentation pattern allows direct determination of the sequence with the de novo sequence. This sequence can be used to match protein sequence databases or to investigate post-translational or chemical modifications. This may provide additional evidence for the identification of proteins performed as above. N- and C-termini
Peptides matched during protein identification should not include predicted N-or C-termini for suitable proteins. This may be the result of an N-or C-terminal peptide that is difficult to identify by the MS (eg too short or too long), after modified post-translation (eg, acetylation of N-terminal) or completely different from the prediction. Post-translational modifications or truncated termino can be identified by closer examination of the data (ie de novo sequencing). Digesting using different protease specificity can also be useful.
Post-translational modifications
While detailed comparisons of MS data with predictions based on a known protein sequence can be used to determine post-translational modification, a targeted approach to data acquisition can also be used. For example, the specific enrichment of phosphopeptides may help identify the location of phosphorylation in a protein. Alternative methods of peptide fragmentation in a mass spectrometer, such as ETD or ECD, may provide complementary sequence information.
Determination of the entire mass
The mass of the entire protein is the sum of the mass of the amino acid residue plus the mass of the water molecule and is adjusted for any post-translational modification. Although the ionized protein is less good than the peptide originating thereof, the protein in the solution may be subject to ESI-MS and its mass is measured with 1 part accuracy in 20,000 or better. This is often enough to confirm the termini (thereby that the mass is measured predicted mass predicted out of sequence) and concludes the presence or absence of many post-translational modifications.
Limitations
Proteolysis does not always result in a set of ready-to-be-analyzed peptides covering all POI sequences. Fragmentation of peptides in mass spectrometers often does not produce ions corresponding to cleavage on any peptide bond. Thus, the sequence inferred for each peptide is not necessarily complete. The standard method of fragmentation does not distinguish between leucine and isoleucine residues because they are isomeric.
Because Edman degradation progresses from N-terminus proteins, it will not work if the N-terminus has been chemically modified (eg by acetylation or Pyroglutamic acid formation). Edman's degradation is generally useless to determine the position of the disulphide bridge. It also requires a peptide count of 1 picomole or more for visible results, making it less sensitive than mass spectrometry.
Predicting from a DNA/RNA circuit
In biology, proteins are produced by messenger messenger RNA (mRNA) with a sequence of proteins derived from the codon sequence in mRNA. The MRNA itself is formed by gene transcription and can be further modified. This process is sufficiently understood to use computer algorithms to automate predictions of protein sequences from DNA sequences, such as from intact gene-DNA-sequencing projects, and has led to the generation of large database sequences of proteins such as UniProt. The predicted protein sequence is an important source for protein identification by mass spectrometry.
Historically, the short protein sequences (10 to 15 residues) determined by the degradation of Edman were re-translated into DNA sequences that could be used as probes or primers to isolate clones of molecules from suitable genes or complementary DNA. The sequence of cloned DNA is then determined and used to infer complete sequence of amino acids from proteins.
Bioinformatics Tool
The bioinformatics tool exists to aid the interpretation of the mass spectrum (see De novo peptide sequencing), to compare or analyze protein sequences (see Sequence analysis), or to search for databases using peptide or protein sequences (see BLAST).
See also
- Proteomics
- DNA Sequencing
- Matthias Mann
- John R. Yates
References
Further reading
Source of the article : Wikipedia
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