Top-Down Proteomics: Ready for Prime Time?

Bifan Chen, Kyle A. Brown, Ziqing Lin, Ying Ge
2017 Analytical Chemistry  
SAMPLE PREPARATION STRATEGIES Often overlooked, sample preparation remains one of the most important and challenging aspects in top-down proteomics. Although MS is a sensitive analytical technique, isotope and charge state distributions of protein ions produced by electrospray ionization (ESI), spreads the signal of a single species over a large m/z range. Thus, signal suppression from salt adducts (i.e. Na + , K + ), detergents, or even coexisting protein species can greatly hamper a top-down
more » ... xperiment. In the section below, common methods for extracting intact proteins, replacing/removing buffer components incompatible with MS, and approaches to decrease sample complexity and to enrich low-abundance proteins will be discussed. Chen et al. Given the complexity and dynamic range of the proteome, enrichment strategies represent an important area of development. Organelle fractionation by differential centrifugation is widely used to isolate and enrich specific sub-proteomes. 46 Nuclear, mitochondrial, membrane, and cytosolic fractions can be collected for global protein analysis and deep proteoform coverage of organelle specific targets. 47 One common example is histone Chen et al. Recently, without a protein precipitation step, Cai et al. developed a MS-compatible serial-SEC (sSEC) strategy that enabled detection of proteins up to 223 kDa using a highresolution Q-TOF mass spectrometer (Figure 2a-b) . 69 By employing a two-dimensional sSEC-RPC approach, more than 4000 unique proteoforms were detected with a 15-fold increase of the proteins above 60 kDa, which greatly outperformed one-dimensional RPC, especially on high MW proteins. 69 Importantly, many of these high MW proteins undetected in one-dimensional RPC, possessed important PTMs (e.g. phosphorylation) (Figure 2c) . Therefore, size-based fractionation prior to other dimensions of separation will continue to play an important role in improving coverage of the intact proteome. Despite its effectiveness, the large amount of sample requirement and labor-intensive offline sample Chen et al.
doi:10.1021/acs.analchem.7b04747 pmid:29161012 pmcid:PMC6138622 fatcat:gq2bxvzsojgwbdvvmll7inhwb4