CPSA Digest 2003

Day 1

Vendor Session - Proteomics Technologies & Applications

Microfluidic HPLC System for Highly Sensitive Peptide Identification by LC-MS

Liquid chromatography coupled to mass spectrometry (LC/MS) is one of the most powerful methods for the analysis of complex peptide and protein mixtures. LC offers a number of benefits including the ability to automate the separation process, direct coupling to the mass spectrometer, 1-dimensional, 2-dimensional and higher separation possibilities, and a choice of a number of separation mechanisms (charge, size, hydrophobicity, affinity). Traditional HPLC systems rely on passive flow splitting to achieve the 100-200 nL/min flow rates required for the most sensitive nanospray ESI mass spectrometers commonly used in proteomics research. However, passive flow splitters suffer from lack of flow rate reproducibility, instability of flow with backpressure changes, and inability to change the flow rate rapidly. These limitations decrease detection sensitivity, adversely affecting the system's ability to identify low-abundance proteins. We will describe the development of a microfluidic HPLC system that provides direct, pulseless control of gradients at separation column flow rates of 20-200 nL/min, without the need for flow splitting. The microfluidic flow control system provides the ability to lower the separation column flow rate rapidly while maintaining the gradient profile. This capability, known as peak parking, provides additional time to run tandem MS on chosen peptides. We will present sensitivity data at various flow rates and the use of peak parking to identify phosphorylated peptides.

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