
The Complete Guide for the Principle and Steps of Protein Sequencing - benniebio
http://www.creative-proteomics.com/services/de-novo-peptides-proteins-sequencing-service.htm
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benniebio
N-terminal sequencing service Almost all of the protein synthesis starts at
the N-terminus, and the N-terminal sequence of proteins has a great influence
on the biological function of the protein as a whole. For example, N-terminal
sequence affects the half-life of proteins, meanwhile, it is associated with
protein subcellular organelles. These are closely related to the function and
stability of proteins. N-terminal sequencing of proteins is helpful to analyze
the high-level structure of proteins and reveal the biological functions of
proteins.

At present, the N-terminal sequencing of proteins is mainly classified into
two major categories: non-mass spectrometry and mass spectrometry. Traditional
non-mass spectrometry includes classical Edman degradation method that takes
advantage of transcription-RT-PCR to get cDNA of corresponding protein, and
then protein sequence will be obtained by reverse counting. Mass spectrometry
is an important method for the accurate mass determination and
characterization of proteins, and a variety of methods and instrumentations
have been developed for its many uses. Its applications include the
identification of proteins and their post-translational modifications, the
elucidation of protein complexes, their subunits and functional interactions,
as well as the global measurement of proteins in proteomics. Each of which has
its own strengths and constraints.

All the above is about the details of protein sequencing which can be applied
for protein identification, also will be helpful for studying the biological
function of the peptide/protein.

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benniebio
Protein sequencing is the practical process of determining the amino acid
sequence of all or part of a protein or peptide, which can be used to identify
the protein or characterize its post-translational modifications. It mainly
refers to the determination of the primary structure of the protein. The
primary structure of the protein comprises the number of polypeptide chains
that make up the protein.The main strategy for protein sequencing is to divide
the polypeptide chain by chemical or enzymatic digestion and then determine
the amino acid residue content and composition.

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benniebio
Protein Sequencing Steps 1\. Cleavage of polypeptide chains. Protein molecules
which is consisted of multiple polypeptide chains must be resolved first.
Several polypeptide chains are linked together by noncovalent bonds, called
oligomeric proteins, such as hemoglobin tetramer, enolase dimer; can be
treated with 8 mol / L urea or 6 mol / L guanidine hydrochloride Separate
polypeptide chains.

2\. Determination of the number of polypeptide chains in the protein molecule.
The number of polypeptide chains can be determined by measuring the
relationship between the number of moles of terminal amino acid residues and
the molecular weight of the protein. 3\. Disulfide bond. Several polypeptide
chains are cross-linked by disulfide bonds and can be treated with excess
beta-mercaptoethanol in the presence of 8 mol / L urea or 6 mol / L guanidine
hydrochloride to reduce the disulfide bond to mercapto. The resulting thiol
group should be protected with an alkylating agent to prevent it from being
reoxidized.

4\. Dertermine each polypeptide chain of the amino acid composition and
caculate the molecular ratio of the amino acid component.

5.Analyzing N-terminal and C-terminal of the polypeptide chain Peptide chain
end group amino acids are divided into two classes: amino-terminal and
Carboxyl-terminal. In the peptide chain amino acid sequence analysis, the most
important is the N-terminal amino acid analysis. N-terminal analysis (Sanger
method; Edman method; DNS-Cl; enzymatic degradation), C-terminal analysis
(hydrazinolysis; enzymatic degradation; lithium borohydride).

6\. The polypeptide chain breaks into multiple peptides. The peptide sample
can be broken into two or more sets of peptide fragments or peptides by two or
more different fracture methods.

7.Determining the amino acid sequence of each peptide.

8\. Determining the order of peptides in the polypeptide chain. The amino acid
sequence of the entire polypeptide chain is interspersed with the overlapping
of the amino acid sequences of two or more sets of peptides.

9.Determine the position of the disulfide bond in the original polypeptide
chain. Generally, pepsin is used to treat the peptide chain without
disconnecting the disulfide bond. And the peptide is separated by two-
dimensional electrophoresis which can analyze and sequence the the peptide
group that may contain the disulfide bond after treatment with formic acid.
Methods were analyzed for peptide comparisons to determine the position of
disulfide bonds.

