High sensitivity amino acid sequence determination. Application to proteins eluted from polyacrylamide gels

Area of Science:

• Biochemistry
• Proteomics
• Analytical Chemistry

Background:

• Determining protein sequences is crucial for understanding protein function and biological processes.
• Traditional sequencing methods often require larger sample quantities and can suffer from significant sample loss.
• Developing sensitive and efficient protein sequencing techniques is essential for modern biological research.

Purpose of the Study:

• To develop an automated solid-phase procedure for determining the amino-terminal amino acid sequence of very small protein quantities.
• To enhance the sensitivity and minimize sample loss during protein sequencing.
• To enable sequencing of proteins directly from polyacrylamide gels.

Main Methods:

• Covalent attachment of protein samples to an inert support to prevent mechanical and physical losses.
• Utilizing a high-sensitivity approach with an initial coupling of phenyl [35S]isothiocyanate followed by unlabeled reagent.
• Identification of radioactive phenylthiohydantoins via autoradiography and two-dimensional thin-layer chromatography.
• Addition of unlabeled phenylthiohydantoin-amino acids as carriers to reduce losses.

Main Results:

• Successful determination of amino-terminal sequences up to 20 residues for protein quantities ranging from 2.5 to 70 pmol.
• The method effectively minimizes sample loss through covalent attachment and carrier addition.
• Demonstrated applicability to proteins eluted from sodium dodecyl sulfate-containing polyacrylamide gels without detergent removal.

Conclusions:

• The described automatic solid-phase procedure offers a highly sensitive and efficient method for protein sequencing.
• This technique is suitable for analyzing minute protein samples, including those directly from gel electrophoresis.
• The method advances the capability for protein sequence analysis in proteomics and biochemical research.