The structural domains of proteins have often been identified through the use of limited proteolysis. In structural genomics studies, it is necessary to carry this out in a high-throughput manner. Here, we constructed a novel high-throughput system, which consists of cell-free protein expression and one-step affinity purification, followed by limited proteolysis using a unique new method, referred to "on beads method". All these steps were carried out on 96-well plate formats and completed in two days, even by manual handling. The merits of the new method versus the conventional one are as follows: (1) experimental times are reduced, (2) the sample preparation for limited proteolysis experiments is simplified, and (3) both protein purification and limited digestion can be performed "in situ" on the same sample plate. This preparation method is therefore suitable for highly automated, proteolytic analyses coupled to mass spectrometry techniques at a micro-scale protein expression level. The resulting protease-resistant fragments were analyzed by MALDI-TOF-MS and protein domains of 34 mouse cDNA products were identified with this system. (C) 2014 Elsevier Inc. All rights reserved.