Sections
Protein Purification
Protein Extraction
Differential Centrifugation
Salting Out
Dialysis
Column Chromatography
Ion-Exchange Chromatography
Anion-Exchange Chromatography
Size Exclusion Chromatography
Affinity Chromatography
Specific Activity
HPLC
Spectrophotometry
Native Gel Electrophoresis
SDS-PAGE
SDS-PAGE Strategies
Isoelectric Focusing
2D-Electrophoresis
Diagonal Electrophoresis
Mass Spectrometry
Mass Spectrum
Tandem Mass Spectrometry
Peptide Mass Fingerprinting
Overview of Direct Protein Sequencing
Amino Acid Hydrolysis
FDNB
Chemical Cleavage of Bonds
Peptidases
Edman Degradation
Edman Degradation Sequenator and Sequencing Data Analysis
Edman Degradation Reaction Efficiency
Ordering Cleaved Fragments
Strategy for Ordering Cleaved Fragments
Indirect Protein Sequencing Via Geneomic Analyses

Concept #1: Edman Degradation

Practice: The peptide Leu—Cys—Arg—Ser—Gln is subject to Edman degradation. Products of the 1st cycle include:

Practice: Suppose you isolated a nonapeptide (9 amino acid residues) from a patient’s blood. Reaction of the nonapeptide with FDNB followed by acid hydrolysis produces a DNP-product with a sulfhydryl R-group, indicating that:

Practice: Treatment of the nonapeptide from the previous problem (1-1) with CNBr produces a tetrapeptide containing the N-terminal amino acid and a pentapeptide. After one round of Edman degradation on the pentapeptide, a product is produced that contained a nonpolar, aliphatic R group, meaning that the pentapeptide has:

Practice: The second and third rounds of Edman degradation on the same pentapeptide from the problem above (1-2) produced products with aliphatic alcohol groups, meaning that the pentapeptide had:

Practice: Hydrazinolysis of the same pentapeptide from the problems above produced modified amino acids & a free α-amino acid with an aromatic-alcohol group. Combining the info from these problems, the pentapeptide is most likely:

Practice: Deduce the entire sequence of the original nonapeptide using the following hint and the information from the previous four practice problems (1-1 through 1-4). Hint: The sequence of the nonapeptide using the one-letter amino acid codes reveals a relevant academic subject.

Practice: A group of peptides that influence nerve transmission in certain parts of the brain has been isolated from normal brain tissue. These peptides are known as opioids, because they bind to specific receptors that also bind opiate drugs, such as morphine and naloxone. Opioids thus mimic some of the properties of opiates. Some researchers consider these peptides to be the brain’s own painkillers. Using the information below, determine the amino acid sequence of the pentapeptide opioid leucine enkephalin. Explain how your structure is consistent with each piece of information below. 

● Complete hydrolysis by 6 M HCl at 110 ˚C followed by amino acid analysis indicated the presence of Gly, Leu, Phe, and Tyr, in a 2:1:1:1 molar ratio. 

● Treatment of the peptide with 1-fluoro-2,4-dinitrobenzene (FDNB) followed by complete hydrolysis and chromatography indicated the presence of the 2,4-dinitrophenyl (DNP) derivative of tyrosine. 

● Complete digestion of the peptide with chymotrypsin followed by chromatography yielded free tyrosine and leucine, plus a tripeptide containing Phe and Gly in a 1:2 ratio.