BISC 330L Biochemistry Spring 2015 USC Lecture 9 Monday Feb 2 2015 Chapter 3 Biochemistry Toolbox Techniques in Protein Analysis Determining Protein Sequence 330 L From last lecture how to identify a protein in a 2D gel Why are amino acid sequences important Identify a protein Can make comparisons to other known proteins Proteomics Reveal evolutionary pathways Search for unusual structural features e g internal repeats Can say something about destination or processing of protein Prepare antibodies allow for further analysis Can make DNA probes Determining amino acid composition Peptide hydrolyzed in 6M HCl at 110oC for 24 h Asx Glx Ion exchange chromatography Identity revealed by elution volume Quantity revealed by Ninhydrin Can also use Fluorescamine Nihydrin reaction with amino acid Fluorescamine reaction with amino acid Protein Sequence Determination Edman Degradation It is useful to know the specific amino acid sequence of a protein Edman Degradation method developed in the 1950s by Pehr Edman to determine protein sequence With genome sequencing this method is not as widely used Protein sequence can be deduced directly from the DNA sequence based on The Genetic Code But it is still used to identify unknown protein in a complex Edman Degradation Edman Degradation Approximately only 30 50 amino acids can be accurately determined To sequence an entire protein small sections have to be determined and then pieced together Start with a protein purified to homogeneity You can use proteases that cleave bonds between specific amino acid pairs Generates a small protein puzzle where the pieces can be put together to determine the order of the fragments Cleavage into small peptides Cleavage by Cyanogen Bromide Cleavage by Trypsin Chemical and Enymatic Cleavage Edman Degradation polypeptide C cleave with protease N N N C N N C N C N C smaller peptides N C C N C C These peptides can then be subjected to Edman degradation process The entire protein sequence can be pieced together like a puzzle only after sequencing at least TWO sets of protease fragments Now let s put the puzzle together DNA sequence Protein sequence What can you do with very long proteins Edman degradation may not work Use genetic code to elucidate a a sequence Only provides nascent protein How to deal with disulfide linked peptides Other approaches to identify proteins I antibodies High affinity and ppt Specific probe structural features Generation of antibodies the hybridoma technology Other approaches to identify proteins II Mass Spectrometry Matrix Assisted Laser Desorption Ionization Time Of Flight MALDI TOF Protein sample is ionized by laser Electrical field accelerates ions toward detector Lightest ions arrive first Time of flight for ions is measured Example of MALDI TOF Mass Spec Analysis End of Lecture 9