Protein Sequencing Procedure If more than one polypeptide chain,separate. Cleave(reduce)disulfide bridges Determine composition of each chain amino acid analysis; Determine N-and C-terminal residues by Edman degradation and C- terminal proteases Cleave each chain into smaller fragments by site-specific proteases and determine the sequence of each chain by the above methods Repeat the above step,using a different cleavage procedure to generate a different set of fragments Reconstruct the sequence of the protein from the sequences of overlapping fragments
Protein Sequencing Procedure If more than one polypeptide chain, separate. Cleave (reduce) disulfide bridges Determine composition of each chain amino acid analysis; Determine N- and C-terminal residues by Edman degradation and Cterminal proteases Cleave each chain into smaller fragments by site-specific proteases and determine the sequence of each chain by the above methods Repeat the above step, using a different cleavage procedure to generate a different set of fragments. Reconstruct the sequence of the protein from the sequences of overlapping fragments
Mass Spectrometry Mass spectrometry is increasingly used in protein identification MS determines the mass-to-charge (m/z)ratio of protein mixtures or peptides (if hydrolyzed by a protease): Evaporate and ionize molecules in a vacuum Separate the ions in space and/or time based on their m/z ratios; Measure different m/z ratios
Mass Spectrometry Mass spectrometry is increasingly used in protein identification MS determines the mass-to-charge (m/z) ratio of protein mixtures or peptides (if hydrolyzed by a protease): Evaporate and ionize molecules in a vacuum Separate the ions in space and/or time based on their m/z ratios; Measure different m/z ratios
Mass Spectrometer Schematic drawing of a mass spectrometer.(1)Highly charged protein droplets are formed by electrostatic dispersion of a protein solution through a glass capillary subjected to a high electric field (electrospray);This step can also be replaced by matrix-assisted desporption ionization (MALDI)(2)Protein droplets are accelerated by electrostatic forces in vacuum;(3)the time it takes for a protein to arrive at the mass spectrometer depends on its m/z.Thus an unknown protein mass can be obtained by comparing its m/z with that of a standard protein. Mass analyzer (1) (2） (3) High voltage Vacuum
Mass Spectrometer Schematic drawing of a mass spectrometer. (1) Highly charged protein droplets are formed by electrostatic dispersion of a protein solution through a glass capillary subjected to a high electric field (electrospray); This step can also be replaced by matrix-assisted desporption ionization (MALDI) (2) Protein droplets are accelerated by electrostatic forces in vacuum; (3) the time it takes for a protein to arrive at the mass spectrometer depends on its m/z. Thus an unknown protein mass can be obtained by comparing its m/z with that of a standard protein. Mass analyzer + + + + + + + + + + (1) (2) (3) High voltage Vacuum
3.3.Secondary Structure in Proteins Any discussion of protein folding and structure must begin with the peptide bond,the fundamental structural unit in all proteins
3.3 • Secondary Structure in Proteins Any discussion of protein folding and structure must begin with the peptide bond, the fundamental structural unit in all proteins
The partial double bond character of the peptide bond
•The partial double bond character of the peptide bond