Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC and ZIC-cHILIC) Provide High Resolution Separation and Increase Sensitivity in Proteome Analysis

Citations Over TimeTop 10% of 2011 papers

Abstract

The complexity of peptide mixtures that are analyzed in proteomics necessitates fractionation by multidimensional separation approaches prior to mass spectrometric analysis. In this work, we introduce and evaluate hydrophilic interaction liquid chromatography (HILIC) based strategies for the separation of complex peptide mixtures. The two zwitterionic HILIC materials (ZIC-HILIC and ZIC-cHILIC) chosen for this work differ in the spatial orientation of the positive and negative charged groups. Online experiments revealed a pH-independent resolving power for the ZIC-cHILIC resin while ZIC-HILIC showed a decrease in resolving power at an acidic pH. Subsequently, we extensively evaluated the performances of ZIC-HILIC and ZIC-cHILIC as first dimension in an off-line two-dimensional liquid chromatography (2D-LC) strategy in combination with reversed phase (RP), with respect to peptide separation efficiency and how the retention time correlates with a number of peptide physicochemical properties. Both resins allowed the identification of more than 20,000 unique peptides corresponding to over 3500 proteins in each experimental condition from a remarkably low (1.5 μg) amount of starting material of HeLa lysate digestion. The resulting data allows the drawing of a comprehensive picture regarding ZIC- and ZIC-cHILIC peptide separation characteristics. Furthermore, the extent of protein identifications observed from such a level of material demonstrates that HILIC can rival or surpass traditional multidimensional strategies employed in proteomics.

Related Papers

• → Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique(2011)1,188 cited
• → Application of hydrophilic interaction chromatography for the analysis of polar contaminants in food and environmental samples(2011)108 cited
• → A detailed evaluation of the advantages and limitations of online RP-LC×HILIC compared to HILIC×RP-LC for phenolic analysis(2023)21 cited
• → Hydrophilic Interaction Liquid Chromatography (HILIC): A Review(2023)2 cited
• [Development of hydrophilic interaction chromatographic hyphenated techniques and their applications].(2008)