Updated project metadata. We present an acetonitrile-based precipitation methodology which 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 to 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 of 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.