Separation methodology to improve proteome coverage depth

So-called 'in-depth proteomics' and its applied separation methodology to improve the proteome coverage depth has become an important issue in mass spectrometric-based proteomics and system-wide cell biology studies. Employing a bottom-up approach and a variety of separation techniques, it allows for identification of proteins with low copy numbers and enables researchers to correlate the number of expressed genes in a cell with the proteome. Here we describe recent advances in this field with emphasis on peptide and protein separation technologies. The discussion is focused both on single injection analyses employing long reversed phase liquid chromatography separations of peptides ('single shot proteomics') and on the combination of orthogonal protein and peptide separation methods to achieve maximum protein coverage. Owing to these improvements, in-depth proteomics has now fully entered the field and is being implemented in an increasing number of laboratories.