Separation methodology to improve proteome coverage depth

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.

Keywords::

• data dependent acquisition
• in-depth proteomics
• orthogonal and multidimensional protein and peptide separation
• single shot proteomics

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• ‘In-depth proteomics’, that is, the attempt to identify all proteins expressed and hence present in a biological system.

• Significant analytical challenges from protein complexity and especially dynamic range, since no protein-amplification technology is available.

• Chromatographic resolution and acquisition speed of the mass spectrometer are maximized to compensate for sample complexity.

• ‘Single-shot proteomics’ to minimize the impact of additional dimensions of protein/peptide separation on instrument time and reproducibility.

• 90% proteome coverage in yeast and 50–60% in human cell culture are made possible by ‘single-shot’ analyses.

• Multi-dimensional protein and peptide separations still add an additional level of analysis depth.

• Re-annotations of genomes are performed on the basis of ‘in-depth proteomics’ studies.