Sensitive detection of peptides in Cytochrome C digest using nano LC with micro Pillar Array Columns (µPACᵀᴹ) and Microsaic 4500 MiD Mass Spectrometer

Liquid chromatography coupled with mass spectrometry is a powerful analytical tool for detection and identification of chemicals in complex mixtures. The Microsaic 4500 MiD® is a miniaturised single quadrupole mass detector designed with the chemist in mind. It retains the performance of a conventional MS system but is considerably smaller and less complex to operate and maintain with a substantially reduced cost of ownership. The 4500 MiD® has been designed with the vacuum system, electronics and computer all inside one box meaning the instrument can be installed in places where no other MS can be easily deployed and no floor pump is needed.

PharmaFluidics’ μPAC™ technology (micro Pillar Array Column) is a unique and novel approach to a chromatographic support structure and builds upon micromachining chromatographic separation beds into silicon, with exceptional properties that result in excellent chromatographic performance with high-resolution and high sensitivity. The inherent high permeability and low ‘on-column’ dispersion obtained by the perfect order of the separation bed makes μPAC™ based chromatography unique in its kind and offers several advantages compared to conventional column technologies (packed beds and monoliths). With a flow rate range between 0.1 and 1.5 μl/min, these columns are ideally suited for a variety of applications where direct interfacing with ESI-MS is needed for sensitive detection and identification of chemicals in complex mixtures.

The compact nature of the Microsaic 4500 MiD® MS system and the combination of low pressure requirements and high separation resolution that PharmaFluidics μPAC™ LC columns can offer have made this combination a perfect fit for performing targeted proteomics studies in an environment where technical resources are limited. Reversed phase separations of cytochrome c tryptic digest are routinely employed for the quality control of HPLC column performance towards proteomics applications. This application note describes the Nano LC-MS analysis of cytochrome C digest using a PharmaFluidics μPAC™ column under various elution programs. The mass spectrometer used was the Microsaic 4500 MiD® alongside the standard UV detector for comparison.