Capillary liquid chromatography coupled with mass spectrometry for analysis of nanogram protein quantities on a wide-pore superficially porous particle column in top-down proteomics

In top-down proteomics experiments, intact protein ions are subjected to gas-phase fragmentation for MS analysis without prior digestion. This approach is used to characterize post-translational modifications and clipped forms of proteins, avoids several inference problems associated with bottom-up proteomics, and is well suited to the study of proteoforms. In the past decade, top-down proteomics has progressed rapidly, taking advantage of MS instrumentation improvements and the efforts of pioneering groups working to improve sample handling and data processing. The potential of this technology has been established through its successful use in a number of important biological studies. However, many challenges remain to be addressed like improving protein separation capabilities such that it might become possible to expand the dynamic range of whole pro-teome analysis, address co-elution and convoluted mass spectral data, and aid final data processing from peak identification to quantification. In this study, we investigated the use of a wide-pore silica-based superficially porous media with a high coverage phenyl bonding, commercially packed into customized capillary columns for the purpose of top-down proteomics. Protein samples of increasing complexity were tested, namely subunit digests of a monoclonal antibody, components of purified histones and proteins extracted from eukaryotic ri-bosomes. High quality mass spectra were obtained from only 100 ng of protein sample while using difluoroacetic acid as an ion pairing agent to improve peak shape and chromatographic resolution. A peak width at half height of about 15 s for a 45 min gradient time was observed on a complex mixture giving an estimated peak capacity close to 100. Most importantly, efficient separations were obtained for highly diverse proteins and there was no need to make method specific adjustments, suggesting this is a highly versatile and easy-to-use setup for top-down proteomics.

Automated summary

In top-down proteomics, intact protein ions are fragmented for MS analysis. This approach has been used to study post-translational modifications and clipped forms of proteins. In this study, a silica-based media with phenyl bonding was used in capillary columns for top-down proteomics, and efficient separations were achieved for diverse proteins.