Depletion of High-Molecular-Mass Proteins for the Identification of Small Proteins and Short Open Reading Frame Encoded Peptides in Cellular Proteomes

Abstract

The identification of small proteins and peptides (below ca. 100-150 amino acids) in complex biological samples is hampered by the dominance of higher-molecular-weight proteins. On the contrary, the increasing knowledge about alternative or short open reading frames creates a need for methods that allow the existence of the corresponding gene products to be proven in proteomics experiments. We present an acetonitrile-based precipitation methodology that depletes the majority of proteins above ca. 15 kDa. Parameters such as depletion mixture composition, pH, and temperature were optimized using a model protein mixture, and the method was evaluated in comparison with the established differential solubility method. The approach was applied to the analysis of the low-molecular-weight proteome of the archaea Methanosarcina mazei by means of LC-MS. The data clearly show a beneficial effect from a reduction of complexity, especially in terms of the quality of MS/MS-based identification of small proteins. This fast, detergent-free method allowed for, with minimal sample manipulation, the successful identification of several not yet identified short open reading frame encoded peptides in M. mazei.