A Novel Nanoflow LC–MS Approach for Bottom-up Proteomics Using the 200 cm long µPAC™ column
Bottom-up proteomics using 50 to 100 µm C18 packed capillaries coupled to high resolution mass spectrometers is used to analyze protein samples from tissues, body fluids or cell lysates. Typically, micrograms of samples are separated in 30 to 240 min nano LC gradients. However, the lack of ease-of-use and reproducibility of nanoflow LC–MS using packed capillaries does not yet allow novice and routine use.
For nanoflow LC–MS for proteomics analysis, the use of micro-chip based pillar array chromatography columns brings significant benefits. In contrast to conventional LC columns that contain randomly packed beads as their stationary phase, micro-chip based pillar array chromatography columns have a separation bed of perfectly ordered and free-standing pillars obtained by lithographic etching of a silicon wafer. The regular mobile phase flow pattern through these micro-chip pillar array columns adds very little dispersion to the overall separation, resulting in better peak resolution, sharper elution peaks and increased sensitivity. The free-standing nature of the pillars also leads to much lower back pressure buildup, and makes it possible to operate longer columns at moderate system pressures.
For bottom-up proteomics, the 200 cm µPAC™ nanoLC column is the separation method of choice.
In this webinar, Dr. Geert van Raemdonck and Gilles Jaouen of PharmaFluidics explained the principles of the 200 cm µPAC™ column and presented high resolution proteomics data using micro-chip based pillar array columns.