Protein Identification

Methods of Determining Amino Acids Composition

• Classic Method: Heat protein at 100oC in concentrated HCl
• Analyze on Ion exchange or HPLC
• Edman degradation: Determine protein concentration from the N-terminal end
• A chromophoric agent binds to the N-terminal of the protein and cleaves the peptide bond
• Detect amino acids via HPLC

• Carboxypeptidase: Binds to the C-terminal of the protein and cleaves the peptide bond
• Detect amino acids via HPLC

• These methods are useful but are limited by the length of the protein.

Protein Detection Techniques

• Chromogenic-Enzyme Assays - change color if the enzyme is present and functional. The rate of color change can inform the researchers as to how much enzyme is present.

• Antibody Assays - an antibody labelled with an enzyme, fluorescent molecule, or radioactive isotope can be used to bind to the protein. The amount of protein in a sample can be detected based on how many antibodies are detected.

• GFP-tagging - Green Fluorescent Protein (GFP) can be used to tag a protein. When the protein is expressed by the cell, GFP, which is fused to the protein, will also "light up" allowing researchers to determine where and how much protein is present.

• Western Blotting/ Immunoblotting - combines gel electrophoresis with protein detection.
• Proteins are run on an SDS-PAGE gel.
• Protein are detected using a primary antibody.
• The primary antibody is detected using a secondary antibody that is conjugated to an enzyme or fluorescent molecule.

• Immunoprecipitation (IP) - Used to detect protein in a mixture of proteins using antibody specificity.
• An antibody is given time to bind to the specific protein of interest that it recognizes.
• An agent that recognizes the antibody is then added to the mixture, which causes the antibody + protein to precipitate out of the solution.

Mass Spectrometry (MS)

• Mass spec analyzers contain:
• 1) An ion source to ionize proteins (MALDI or electrospray);
• 2) Mass analyzer which separates proteins based on their m/z ratio (TOF or ion traps);
• 3) Strike detector which tells the relative abundance of each ion;
• 4) Computerized data system to acquire, store and process data.
• Mass spec is highly sensitive (can detect down to 1x10-15 mol of certain proteins) and can easily distinguish between two very similar proteins.
• Mass spec gives researchers a RELATIVE amount of protein, not an absolute amount.