Protein Sequence Analysis

Protein Sequence Analysis is the process of subjecting a protein or peptide sequence to one of a wide range of analytical methods to study its features, function, structure, or evolution. Many functional characteristics of proteins can be directly obtained by analyzing their sequences. For example, a hydrophobicity map can inform the prediction of transmembrane helices.

Protein sequencing is mainly relying on chemical or enzymatic digestion methods to separate peptides and detect the amount and composition of amino acid residues. Currently, the so-called protein sequencing refers to the detection of proteins' primary structure, which contains the number of polypeptide chains in proteins. Polypeptides and proteins can be used equally in many cases. Amino acid sequence of polypeptides is the biological function of proteins.

Sequencing Steps

• Splitting polypeptide chain
• Detecting the number of polypeptide in protein moleculars: by detecting the relationship between the number of moles of amino acid residues and protein molecular weight
• Breaking disulfide bonds
• Detecting the amino acid composition of polypeptide chains and calculating the molecular ratio of amino acid composition
• Sequencing N-terminal and C-terminal of polypeptide chains
• Breaking polypeptide samples into two or more sets of peptides or peptide fragments and then separate them
• Determining the amino acid sequencing of each peptide
• Determining the sequence of peptide fragments in polypeptide chains
• Determining the position of disulfide bonds in the original polypeptide chain

Sequencing Techniques

• N-terminal determination
  Almost all protein synthesis starts at the N-terminal. Sequencing analysis of the N-terminal of the protein is beneficial to help analyze the high-level structure of the protein and reveal the biological function of the protein. At present, there are two main categories for protein N-terminal sequencing, one is the classic Edman degradation method, and the other is mass spectrometry techniques.

• N-terminal Edman degradation
  Proteins are first modified with phenylisothiocyanate. Subsequently, the derivatized terminal amino acid is removed by acid cleavage in a form of phenylthiohydantoin derivative and a new α-amino group on the next amino acid is now available for the next round of reaction with PITC. Protein sequence is therefore being analyzed in through a serious reaction of PITC addition, and cleavage of one PTH at each time for analysis.
• Mass spectrometry
  Mass spectrometry is a highly efficient method for the accurate mass determination and characterization of proteins. Two mainly used mass spectrometers are electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI), respectively. Basically, proteins are pretreated with digestive enzymes to be digested into small fragments, which are analyzed by mass spectrometers.

• C-terminal determination
  Currently there is no standard chemical method to sequence protein C-terminus, and  LCMS is often a preferred method in protein C-terminal determination. C-terminal sequence analysis is used for direct confirmation of the C-terminal sequence of native and expressed proteins, for detection and characterization of protein processing at the C-terminus and for identification of post-translational proteolytic cleavages.

Applications

• Analysis of amino acid composition of unknown proteins
• Analysis of physical and chemical properties of amino acids: amino acid composition, pI, MW, extinction coefficient, hydrophilic / hydrophobic

Releated Products

Mass Spectrometer

A mass spectrometer is any device that produces a mass spectrum read-out by measuring the individual mass spectra in a sample. It detects the speed at which positively charged ions move through a vacuum chamber toward a negatively charged plate. Mass spectrometers are commonly used in life science research to analyze peptides, amino acids, and proteins. They are also used to facilitate DNA sequencing and analyze intact viruses.

Fluorometer

Fluorometers are routinely used in molecular and cell biology laboratories to detect and quantify fluorescent light signals emitted at different wavelengths. Appropriate filtering by fluorescence instrumentation allows detection of fluorescent signals of different emission wavelengths. Fluorometers with advanced detection capabilities also detect signals from fluorescent resonance energy transfer (FRET) experiments.

Chromatography System

Chromatography systems include all the equipment needed for sample separation, a power source and program software. Important considerations when selecting a chromatography system are the chemical and physical properties of the substance such as chemical reactivity, size, mass and shape and requirements of the laboratory, such as flow process, validation requirements and sample tracking.

STEMart provides you with a variety of protein sequence analysis equipment or consumables to meet your various R&D and application needs. If you have any questions or requirements for sequence analysis, please feel free to contact us.