Protein analysis Tools for protein analysis Progress in science depends on new techniques, new discoveries and new ideas, probably in that order Sydney Brenner 04/25/2023 by TDLV Why we analyse proteins? • Explore how amino acid sequences specify the structure of proteins • How proteins bind to substrates and other molecules to perform their functions • Understand the function of the protein based on its structure 04/25/2023 by TDLV Main Steps in Protein Analysis Extraction of proteins Purification of the protein Structural Characterization of the protein 04/25/2023 by TDLV Protein Characterization • Characterization of proteins and peptides: different processes: Determining the Amino Acid Composition • finding out the amino acids that make up the protein and their number Determining the Amino Acid Sequence • finding out the sequence of amino acids of the proteins in their order Determining the Molecular mass of the Protein 04/25/2023 by TDLV Determination of Amino Acid Composition • peptide  hydrolyzed  amino acids by heating in 6M HCl at 110ºC for 24 hrs • The amino acids  separated by HPLC – measured by reacting them with ninhydrin Alpha Amino acids  intense blue color , imino acids (proline, hydroproline)  yellow color – The concentration of a single amino acid is proportional to the light absorbance of the solution after adding ninhydrin – The elution profile of the amino acids is obtained – The identity of the amino acid by TDLVis revealed by its elution volume 04/25/2023 04/25/2023 by TDLV Determination of protein Molecular Mass by Mass Spectrometry • • Mass Spectrometry: include an ionization source, a mass analyzer and a detector The ion hits the mass analyzer The time the ions take in flight is recorded by triggering a laser pulse It depends on one principle – The smaller ions will fly faster and hit the detector first then the larger ions reach The ions hit the detector in order by mass 04/25/2023 by TDLV Chromatography for protein purification 04/25/2023 by TDLV Chromatography-3 matrices 04/25/2023 by TDLV Protein purification by chromatography 04/25/2023 by TDLV 10 Genetically-engineered tags provide an easy way to purify proteins - antigenic determinant, or epitope, recognized by a specific antibody - short string of histidines (His-tag) binds to an affinity column containing immobilized nickel ions - Glutathione S-transferase (GST) fusion protein binds to an affinity column containing glutathione 04/25/2023 by TDLV 11 Purification of protein complexes by using a GST-tagged fusion protein 04/25/2023 by TDLV 12 Protein analysis  SDS PAGE ( sodium dodecyl sulfate polyacrylamide-gel electrophoresis  Protein separation 04/25/2023 by TDLV 13 SDS PAGE 04/25/2023 by TDLV 14 Separation of proteins by isoelectric point 04/25/2023 by TDLV 15 Specific proteins can be detected by Western blotting 04/25/2023 by TDLV 16 The diffraction of X-ray by protein crystals can reveal a protein’s exact structure 04/25/2023 by TDLV 17 Determing Primary Structure of an Entire Protein