Combined usage of cascade affinity fractionation and LC-MS/MS for the proteomics of adult mouse testis

In this report, the proteomics of adult mouse testis were analyzed by the combined usage of cascade affinity fractionation and LC-MS/MS. The differences between the selected affinity ligands in size, shape, structure, and biochemical characteristics, result in each ligand exhibiting a specific affinity to some protein groups. Therefore, a cascade composition of different ligands can be applied to the fractionation of complex tissue proteins. Ultimately, the fractions collected from cascade affinity fractionation were analyzed by LC-MS/MS, which resulted in high confidence identification of a total of 1378 non-redundant mouse testis protein groups, over 2.6 times as many proteins as were detected in the un-fractionated sample (526). All detected proteins were bioinformatically categorized according to their physicochemical characteristics (such as relative molecular mass, pI, grand average hydrophobicity value, and transmembrane helices), subcellular location, and function annotation. This approach highlighted the sensitivity of this method to a wide variety of protein classes. Utilizing a combination of cascade affinity fractionation and LC-MS/MS, we have established the largest proteomic database for adult mouse testis at the present time.