Facilitating Phosphopeptide Analysis Using the Agilent HPLC Phosphochip

The analysis of phosphopeptides can be particularly challenging since they are mostly present at very low concentrations in a complex proteolytic sample. Selective enrichment strategies have been introduced to capture phosphopeptides from the sample prior to MS analysis, which can enhance detection capabilities. One of the more successful phosphopeptide enrichment techniques is based on the selective capture of phosphopeptides using titanium dioxide (TiO2) packing material. Although successful, these enrichment strategies still face challenges with regards to automation, robustness, reliability and reproducibility. The commercially available Agilent HPLC-Chip/MS provides a platform that can integrate an enrichment column, analytical column, associated connection capillaries, and a nanospray emitter directly on a single, small, reusable microfluidic chip. It also provides an easy to use, robust and reliable nanospray LC/MS platform when compared to conventional nanocolumn LC/MS technology. Integration of enrichment columns with different stationary phase materials on the chip can be used to selectively capture phosphopeptides during the sample loading process. In this Application Note, we will describe the design and performance of an HPLC-Chip with a hybrid TiO2 and C18 enrichment column for selective phosphopeptide enrichment and analysis. Authors