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: Strategy For Ordering Cleaved Fragments: Steps #1 & 2

Concept #2: Strategy For Ordering Cleaved Fragments: Step #3

Concept #3: Strategy For Ordering Cleaved Fragments: Steps #4 & 5

Practice: A sample of an unknown peptide was divided into two aliquots. One aliquot was treated with trypsin; the other was treated with cyanogen bromide. Given the following sequences of the resulting peptide fragments, deduce the sequence of the original peptide.

Trypsin treatment:                                                                         Cyanogen bromide treatment:                 
Asn—Thr—Trp—Met—Ile—Lys                                             Gln—Phe

Gly—Tyr—Met—Gln—Phe                                                     Val—Leu—Gly—Met

Val—Leu—Gly—Met—Ser—Arg                                            Ile—Lys—Gly—Tyr—Met

                                                                                                Ser—Arg—Asn—Thr—Trp—Met

Sequence: 

____-____-____-____-____-____-____-____-____-____-____-____-____-____-____-____-____

Practice: A peptide with 31 amino acid residues is independently treated with trypsin to give four fragments and separately treated with chymotrypsin to give six fragments (see chart below). FDNB treatment followed by amino acid hydrolysis resulted in DNP-Met and free amino acids. Identify the sequence of the 31 amino acid residues in the original unfragmented protein using one-letter amino acid codes.

Practice: The sequence of kassinin, a tachykinin dodecapeptide from the African frog Kassina senegalensis, was determined. A single round of Edman degradation identifies Asp as the N-terminus. A 2nd sample of the peptide is treated with chymotrypsin, releasing two fragments with the following amino acid compositions: fragment 1 (G, T, M, V) and fragment 2 (D2, Q, K, F, P, S, V). Next, a 3rd sample of peptide is treated with trypsin, which results in two fragments with the following amino acid compositions: fragment 3 (D, P, K, V) and fragment 4 (D, Q, G, T, M, F, S, V). A 4th sample was treated with CNBr, but the dodecapeptide was not cleaved.  A 5 th sample treated with elastase yields a single Gly residue & three fragments—fragment 5 (T, M), fragment 6 (D, K, P, S, V), and fragment 7, which was sequenced as: D—Q—F—V. What is the sequence of the dodecapeptide? 

Hint: Elastase cleaves C-terminal side of small neutral residues: G, A, V, L, I & S.

Sequence: _____-_____-_____-_____-_____-_____-_____-_____-_____-_____-_____-_____