Protein biomarker discovery is an area of considerable current interest in biology and medicine.
The implementation of proteomics technology in the field of protein biomarker discovery has expanded in the past few years to enable the identification of biomarkers from a variety of biological samples including cell lysates, tissue samples, serum, plasma and urine (for a review refer to Gao, J. et al. (2005) Methods. 35, 291-302).
One method utilized for the discovery of protein markers includes the use of trypsin digestion of target proteins followed by the analysis of the resulting peptide fragments by mass spectrometry. Typically targeted to biomarkers related to a certain disease stages, this methodology can be expanded to the discovery of biomarkers in research in unrelated fields.
Examples
Objective: As proof of concept use normal C57BL/6 mice maintained under a regular 24-hour light and dark cycles to demonstrate changes in protein abundance in blood.
Design: Plasma was collected from mice at various time points in a 24-hour cycle. Samples were pre-fractioned by column chromatography and were subsequently analyzed on tricine gels. Silver staining was performed and protein bands were excised and digested with trypsin. Sequencing data generated by MS/MS were searched against murine database using SEQUEST.
Objective: Using two-dimensional gel electrophoresis and electrospray ionization mass spectrometry, this study’s aim was to investigate protein expression changes in rabbit cortex induced by chronic exercise.
Design: Kidneys were obtained from New Zealand rabbits confined to pens or trained on a treadmill for 60 minutes a day over a 12-week period. Total protein was isolated and analyzed by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie® blue. Differential protein spots were excised and digested with trypsin and peptides were sequenced by electrospray mass spectrometry.
