Actin-myosin interactions by affinity column chromatography, ELISA, and tandem mass spectrometry

Candidate: Arnold Au
Supervisor: Dr. John Marshall

ABSTRACT

Protein protein interactions are the basis of biological functions, playing a wide range of roles in cellular processes as well as intercellular communication. One of the most abundant and well studied proteins is actin. Actin is a vital protein found in virtually all eukaryotic cells as it performs key roles in the cytoskeleton, intracellular trafficking and cellular locomotion. Myosin is one of the most well studied interactors of actin and possesses prominent actin binding sites that makes an ideal target for studying protein protein interaction alongside actin. ß-Actin and myosin were isolated from RAW 264.7 cell homogenates using several different methods, namely co-immunoprecipitation, affinity purification, enzyme linked immunosorbent assay and direct on-bead digestion. Several methods were used to detect and identify the proteins including western blotting, dot blot, enzymatic (colorimetric and chemiluminescent), nano electrospray liquid chromatography tandem mass spectrometry and enzyme linked immuno mass spectrometric assay. Mass spectra were analyzed by X!TANDEM and SEQUEST peptide search algorithms for protein identification and redundant MS/MS spectra were filtered out with an SQL Server system and R statistical analysis was used to perform Chi Square (X2) analysis of frequency counts. Counts of peptide spectra matches were used as a measure and indicator of protein identifications. The different protein isolation methods all agreed with each other on beta-actin purification, while mass spectrometry was able to provide additional information via identification of gamma actin isoform and several myosin isoforms (I - XIV). Limits of detection were estimated for chemiluminescent and enzyme linked immuno mass spectrometric assay, reaching down to actin detection in 1.99μg of RAW264.7 cell homogenate. With chemiluminescence enzyme linked immunosorbent assays providing the highest sensitivity and mass spectrometry providing the widest range in protein identification, this model of actin-myosin interactions can be applied to the study of any protein-protein interaction and the pros and cons of each protein detection assay.