Hamish D. Morrison

Analysis of dilute peptide samples by capillary zone electrophoresis

We report a method for the analysis of dilute peptide solutions by capillary zone electrophoresis. The procedure is based on an electrophoretic concentration step of the applied peptide solution in the capillary (stacking) prior to separation, thus allowing the application of increased sample volumes without a breakdown in resolution. Given a constant configuration of the hardware, the method permits the analysis of peptide solutions of an at least 5 times lower concentration than previously possible. The method was applied to the direct analysis of peptide samples separated by narrow-bore reversed-phase high-performance liquid chromatography for high-sensitivity peptide-sequence analysis.

Identification of the sites in myelin basic protein that are phosphorylated by meiosis-activated protein kinase p44mpk

Myelin basic protein serves as a convenient substrate for detection of a 44 kDa protein‐serine/threonine kinase (p44 mpk ) that is activated near the time of germinal vesicle breakdown in maturing echinoderm and amphibian oocytes. In vitro phosphorylation by purified p44 mpk from sea star oocytes was primarily on threonine residues on a single tryptic peptide of bovine brain myelin basic protein. Amino acid composition analysis of the isolated posphopeptide revealed that it was rich in proline residues. Automated solid‐phase sequencing by Edman degradation identified the major site as Thr‐97 in the sequence NIVTPRTPPPSQGK, which corresponds to residues 91–104 in bovine brain myelin basic protein. Thr‐94 was also phosphorylated by p44 mpk to a very minor extent.

Synthesis, Evaluation and Application of a Panel of Novel Reagents for Stepwise Degradation of Polypeptides

The Edman degradation (Edman, 1949) has been the most successful, general and widely used technique for the determination of the amino acid sequence of proteins and peptides. As a benefit of this distinction, over the last four decades the method has been refined to a high degree of perfection. Nevertheless, sequencing with phenyl isothiocyanate (PITC)† suffers from a few practical limitations. First, the extinction coefficient of the phenylthiohydantoins (PTH’s) limits sequencing sensitivity. Currently, routine sequencing in most laboratories requires low picomole amounts of sample applied to the sequencer. Second, UV-absorbing products which may co-elute with PTH’s during high performance liquid chromatography (HPLC) separation have a tendency to obscure the specific PTH signals during high sensitivity sequencing. Third, with the exception of select cases (Wettenhall et al, 1991; Meyer et al, 1990, 1991; Aebersold et al, 1991; Gooley et al, 1991; Pisano et al, 1993), modified and unnatural amino acids of known structure are difficult to identify and de-novo characterization of such residues by UV absorbance detection alone is extremely difficult.