Authors
Ben-Bo Gao, Xiaohong Chen, and Edward P. Feener
Summary
Mass spectrometry-based proteomics is widely used for the de novo identification of proteins from body fluids (1). The prefractionation of complex mixtures prior to tandem mass spectrometry can dramatically improve sensitivity for low abundance proteins. A variety of protocols involving reverse phase or ion exchange chromatography, affinity enrichment methods, and in gel electrophoresis has been utilized to prefractionate these complex mixtures. Protein fractionation by one-dimensional sodium dodecylsulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE) provides high resolution, concentrates individual proteins and their resultant tryptic peptide a limited number of fractions, provides an estimate protein molecular weight, and can be used to generate a limited number of fractions that includes the entire sample. We have used preparative 1D SDS-PAGE to fractionate individual vitreous samples into 60 to 70 fractions, processed individual fractions to isolate tryptic digests, and performed sequential tandem mass spectrometric analysis using a LTQ Linear trap mass spectrometer. We developed PHP-MySQL database based software to compile, organize, and filter the data generated from each fraction from each sample and compile results a large number of independent samples into a single table. The protocols that we have developed for proteomic data acquisition and bioinformatic analysis are described.Further details
The protocol was published on Protocol Exchange in 2007. To see the entire protocol, click on the source link.Advertisement
Stats
- Recommendations n/a n/a positive of 0 vote(s)
- Views 292
- Comments 0