protein sequencing

Summary

Protein sequencing can be used to: (1) identify proteins; (2) characterize post-translational modifications of proteins. (3) Analyze the relationship between protein primary structure and function.

Operation method

Edman degradation

Principle

The main methods are mass spectrometry, sequencing using protein sequencers and indirect sequencing using protein corresponding to DNA or mRNA. Traditional protein sequencing experiments generally include the following steps: 1. Unbundling and separation of peptide chains; 2. Determination of the number of polypeptide chains in the protein molecule; 3. Disulfide bond breakage; 4. Determination of the amino acid composition of each polypeptide chain and calculation of the molecular ratios of the amino acid components; 5. Determination of the N-terminal and C-terminal ends; 6. Polypeptide chain breakage; 7. Determination of the amino acid sequence of each peptide; 8. Determination of the order of peptides in the polypeptide chain; 9. Determination of the position of peptides in the polypeptide chain. Determination of the order of the peptides in the polypeptide chain; 9. Determination of the position of the disulfide bond in the original polypeptide chain.

Materials and Instruments

Protein samples
Urea Guanidine hydrochloride Mercaptoethanol
Protein sequencer

Move

1 Splitting of polypeptide chains. Protein molecules composed of multiple polypeptide chains must first be split, generally 8 mol/L urea or 6 mol/L guanidine hydrochloride treatment of proteins to separate the polypeptide chains of proteins.

2. Determine the number of polypeptide chains in the protein molecule. The number of polypeptide chains can be determined by measuring the relationship between the molar number of terminal amino acid residues and the molecular weight of the protein.

3. Disulfide bond breaking. Several polypeptide chains cross-linked together by disulfide bonds can be treated with excess mercaptoethanol in the presence of 8 mol/L urea or 6 mol/L guanidine hydrochloride to reduce the disulfide bonds to sulfhydryl groups, and then the generated sulfhydryl groups can be protected with iodoacetic acid (alkylating reagent) to prevent it from being reoxidized. The unwound peptide chain can be separated by chromatography or electrophoresis.

4. The amino acid composition of each peptide chain is determined and the molecular ratio of the amino acid components is calculated. This can be used to facilitate the detection of errors in the sequencing process or to distinguish ambiguous results. Knowledge of certain amino acid frequencies can also be used to select proteases for protein digestion. Low levels of non-standard amino acids (e.g., n-leucine) incorrectly incorporated into proteins can also be identified.

(1) Hydrolysis. Hydrolysis is performed by heating protein samples in 6 mol/L hydrochloric acid to 100-110°C for 24 hours or longer. Proteins with many bulky hydrophobic groups may require longer heating times. Some amino acids (serine, threonine, tyrosine, tryptophan, glutamine and cysteine) may be degraded. To address this issue, Biochemistry Online suggests heating samples for different times, analyzing each resulting solution, and extrapolating back to zero hydrolysis time. Rastell suggests using various reagents to prevent or minimize degradation, such as thiol reagents or phenol to protect tryptophan and tyrosine from chlorine and to pre-oxidize cysteine. He also recommends measuring the amount of ammonia released to determine the extent of amide hydrolysis.

(2) Separation and quantification. Amino acids can be separated by ion exchange chromatography and then detected by derivatization. Another method is to derivatize the amino acids and then separate them by reversed-phase HPLC.

Ion exchange chromatography is performed using sulfonated polystyrene as a matrix. Amino acids are added to an acid solution and a buffer that stabilizes the increased pH is passed through the exchange column. The amino acids were eluted when the pH reached their respective isoelectric points. Once the amino acids are separated, their respective amounts are determined by adding reagents that will form colored derivatives. If the amount of amino acids exceeds 10 nmol, ninhydrin can be used, which reacts with proline in a yellow color and occurs in a bright purple color with the other amino acids. The concentration of the amino acid is proportional to the absorbance of the resulting solution. Very small amounts or less than 10 pmol of amino acids can be used to form fluorescent derivatives using reagents such as o-phthalaldehyde (OPA) or fluorescent amines.

Pre-column derivatization can be performed using Edman reagents to produce derivatives detectable by UV light. Higher sensitivity can be obtained by using reagents that produce fluorescent derivatives. Derivatized amino acids are subjected to reverse phase chromatography, typically using a C8 or C18 silicon dioxide column and an optimized elution gradient. The eluted amino acids are detected using a UV or fluorescence detector and the peak areas are compared to those of the derivatized standards to quantify each amino acid in the sample.

5. Analyze the N-terminus and C-terminus of the peptide chain.

(1) N-terminal analysis method: (2,4-dinitrofluorobenzene (DNFB) method; phenyl isothiocyanate (PITC) method; dansyl chloride (DNS) method; aminopeptidase method);

(2) C-terminal analysis (hydrazinolysis method; enzymatic degradation method; lithium borohydride method).

6. The polypeptide chain breaks into multiple peptide segments.

Breaking down the peptide chain can be carried out by endopeptidases such as trypsin or pepsin or by chemical reagents such as cyanogen bromide. Different enzymes produce different cleavage patterns, and the overlap between fragments can be used to construct the overall sequence.

7. Determination of the amino acid sequence of each peptide fragment

Peptides to be sequenced are adsorbed onto a solid surface (a common substrate is glass fiber coated with polystyrene (a cationic polymer)), and phenyl isothiocyanate (PITC) is added to the adsorbed peptide along with a mildly alkaline buffer solution of 12% trimethylamine to react with the amine groups of the N-terminal amino acids. The terminal amino acids can then be selectively isolated by the addition of anhydrous acid. The derivatives are then isomerized to give substituted phenylglycolide urea, which can be washed off and identified by chromatography, and the cycle can be repeated. The efficiency of each step is about 98%, permitting reliable identification of about 50 amino acids.

Sequence determination can also be done directly with a protein sequence analyzer. Samples of proteins or peptides are immobilized in the reaction vessel of the protein sequencer and subjected to Edman degradation. Each cycle releases and derives an amino acid from the N-terminus of the protein or peptide, and the released amino acid derivatives are then identified by HPLC. The sequencing process is repeated for the entire peptide until the entire measurable sequence or a predetermined number of cycles are established.

8. Determine the order of the peptides in the polypeptide chain.

The amino acid order of the entire polypeptide chain is pieced together using two or more sets of peptides whose amino acid sequences overlap with each other.

9. Determine the position of disulfide bonds in the original polypeptide chain

According to the known amino acid order, select the appropriate specific protein hydrolase (usually pepsin) to partially hydrolyze the protein without opening the disulfide bond, and then separate the peptides by bidirectional electrophoresis, and then analyze the composition and order of the peptides that may contain disulfide bonds after treatment with peroxyformic acid, and then compare with the peptides analyzed by other methods to determine the position of the disulfide bond.

Caveat

1. The purity of the sample should not be too low, and experience suggests that proteins with a purity greater than 90% are suitable for sequencing reactions.

2. N-terminal closed or glycosylated proteins are difficult to perform Edman reaction and cannot be sequenced.

Common Problems

Other protein sequencing methods are:

1. Short protein sequences (10 to 15 residues), which can be determined by Edman degradation, are translated into DNA sequences that are used as probes or primers to isolate molecular clones of the corresponding gene or complementary DNA. The sequence of the cloned DNA is then determined and used to infer the complete amino acid sequence of the protein.

2. De novo sequencing, also known as de novo protein sequencing. This technique is used to infer the amino acid sequence based on the mass difference between a series of regular fragment ions produced by the collision of a peptide with an inert gas. We can infer the amino acid sequence based on the y and b ions at the peptide bond breaks, as well as post-translational modifications. de novo sequencing has an outstanding advantage that cannot be achieved by traditional mass spectrometry sequencing, that is, it sequences unknown proteins from scratch without relying on any protein database.

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