Abstract
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.
Highlights
-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
Two principal workflows, representing different separation ‘philosophies’, are frequently applied: (i) addition of further dimensions of separation on the intact protein level, for example, according to apparent protein molecular weight by SDS-PAGE, to isoelectric point by electrophoretic separation (OFFGEL electrophoresis, which is a free-flow isoelectric focusing technique that separates and recovers proteins directly in solution for further analysis [29,30]), or to hydrodynamic radius by size-exclusion chromatography [10] before hydrolysis with endopeptidases; (ii) hydrolysis of the protein sample in solution and subsequent application of additional separations on the peptide level, usually various chromatographic workflows to prefractionate the resulting highly complex peptide mixtures before these are submitted to the nanoflow RP-LC front end of the mass spectrometer
When peptides derived from various molecular weight regions are separated with a capillary column packed with RP material of smaller particle size and operated by ultrahigh pressure liquid chromatography pumps [33], the analysis depth of a sample increases dramatically, and up to 8000 proteins from less than 100 mg of sample material from a human cancer cell line can be identified on a routine basis [34]
Summary
To cite this article: Christof Lenz & Henning Urlaub (2014) Separation methodology to improve proteome coverage depth, Expert Review of Proteomics, 11:4, 409-414, DOI: 10.1586/14789450.2014.919862 To link to this article: https://doi.org/10.1586/14789450.2014.919862 Full Terms & Conditions of access and use can be found at https://informahealthcare.com/action/journalInformation?journalCode=ieru20
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