Combining offline high performance liquid chromatography fractionation of peptides and intact proteins to enhance proteome coverage in bottom-up proteomics

Abstract

Offline peptide separation (PS) using high-performance liquid chromatography (HPLC) is currently used to enhance liquid chromatography–tandem mass spectrometry (LC–MS/MS) detection of proteins. In search of more effective methods for enhancing MS proteome coverage, we developed a robust method for intact protein separation (IPS), an alternative first-dimension separation technique, and explored additional benefits that it offers. Comparing IPS to the traditional PS method, we found that both enhance detection of unique protein IDs to a similar magnitude, though in diverse ways. IPS was especially effective in serum, which has a small number of extremely high abundance proteins. PS was more effective in tissues with fewer dominating high-abundance proteins and was more effective in enhancing detection of post-translational modifications (PTMs). Combining the IPS and PS methods together (IPS+PS) was especially beneficial, enhancing proteome detection more than either method could independently. The comparison of IPS+PS versus six PS fractionation pools increased total number of proteins IDs by nearly double, while also significantly increasing number of unique peptides detected per protein, percent peptide sequence coverage of each protein, and detection of PTMs. This IPS+PS combined method requires fewer LC-MS/MS runs than current PS methods would need to obtain similar improvements in proteome detection, and it is robust, time- and cost-effective, and generally applicable to various tissue and sample types.