Valeja et al. (2015) combine two methods to prepare intact proteins for mass spectrometric evaluation, creating a multi-dimensional liquid chromatography (MDLC) workflow.1 The new workflow comprises protein separation by ion exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC) with reverse phase chromatographic (RPC) fractionation. The final protocol, described by the authors as 3-D IEF HIC RPC, gives better performance than 2-D IEC and shows potential for deep proteome characterization in top-down approaches.
Top-down proteomics, in which researchers characterize intact proteins, is a better tool for biomarker discovery than approaches that evaluate proteolytically digested samples. Examining the intact proteins allows characterization at the proteoform level for disease-specific changes in structure, including post-translational modification. However, preparing samples for top-down mass spectrometric proteomic characterization is challenging due to the complexity of the proteome under analysis. For this reason, researchers often use MDLC for efficient, high-resolution separation.
Valeja et al. optimized the combined workflow with standard protein solutions, including myoglobin and ovalbumin, and with extracts from lyzed human embryonic kidney (HEK) 293 cells. They coupled mixed-bed IEC with HIC, in conjunction with RPC, using an EASY-nLC 1000 liquid chromatography system (Thermo Scientific). The researchers used ammonium tartrate as the salt for HIC, having recently confirmed it as compatible with subsequent mass spectrometric protein evaluation. The team then examined the protein fractions by top-down proteomic analysis on a Q Exactive Orbitrap mass spectrometer, collecting data with Xcalibur software revision 2.2 (both Thermo Scientific). They searched data against the National Center for Biotechnology Information human database to identify proteins.
First, the researchers demonstrated orthogonality between IEC and HIC, showing that the two methods could perform well in conjunction. They then examined the logical order for each technique during the analytical workflow, considering potential discordances and compatibilities between buffer use, operating conditions and washout solutions that could affect final performance. Finally, Valeja et al. compared data arising from intact protein separation and analysis by 2-D (IEC RPC) and 3-D (IEC HIC RPC) HEK 293 lysate sample preparation. They identified 640 proteins with the 3-D workflow and only 47 from the 2-D approach.
The 3-D approach performed better for intact protein analysis than the 2-D sample preparation, thus showing great potential for biomarker discovery. However, Valeja et al. suggest that further improvements are needed. The enhanced chromatographic separation does result in an increase in proteins identified, but the team notes that two areas need improvement:
- Optimization for detection of higher–molecular weight analytes between 30 kDa and 250 kDa
- Boosting overall protein recovery
They also suggest increasing analytical speed by managing the number of fractions selected for assessment, although this might compromise the depth of data collected.
1. Valeja, S.G., et al. (2015) “Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics,” Analytical Chemistry, 87(10) (pp. 5363–5371), doi:10.1021/acs.analchem.5b00657.
Post Author: Amanda Maxwell. Amanda is a freelance science writer and digital space explorer with a passionate curiosity for science and technology. She enjoys translating complex theories and subjects creatively into everyday language for all audiences. Equipped with a bachelor’s degree in veterinary medicine and a PhD in protein chemistry/small animal critical care nutrition, she brings clinical experience and practical research oversight into her writing.