Capillary LC-MS Methods for Trace Level Peptide Detection and its Application for Monitoring Neuropeptides In Vivo.

Abstract

Capillary liquid chromatography – mass spectrometry (cLC-MS) is an emerging technique for detecting low abundance peptides. Its practicality in in vivo neuropeptide monitoring from microdialysate was limited to 10 out of more than 200 mammalian neuropeptides due to the slow analysis rate and uneven sensitivity for different peptides. The slow speed is not favorable for in vivo study which generates many samples that degrade during storage. The uneven sensitivity results in high limit of detection (LOD) for some neuropeptides, making them undetectable in dialysate. In this dissertation, cLC-MS’s performance was improved in these two aspects. The rate limiting step of cLC-MS was the long sample injection process due to high back pressure using a 25 μm bore column. In chapter 2 a method utilizing large particle and 75 μm bore column was developed to enhance injection speed. The method reached 4 min/sample throughput compared to previously reported throughput at 20-30 min/sample with similar LOD. This was achieved by rapidly injecting sample under 14 μL/min and eluting slowly under 100 nL/min for nanoESI sensitivity. This method was applied for monitoring enkephalins from rat globus pallidus. In chapter 3, acetonitrile (ACN) was used as organic additive in sample to increase neuropeptide solubility and decrease nonspecific adsorption. Adding ACN to optimal concentration improved LOD to low pM range (0.1-2 pM) for all 10 neuropeptides tested in the study. In addition, modification of the microdialysis probe using polyethyleneimine (PEI) increased sampling recovery by reducing electrostatic interaction between neuropeptides and microdialysis catheter. This method was applied for monitoring intact orexins from rat arcuate nucleus. Application of cLC-MS can be extended to quantifying low abundance proteins using a peptide fragment produced from protease digestion. In Chapter 4 a signature peptide method for brain derived neurotrophic factor (BDNF) was developed. For BDNF, chymotrypsin produced higher signature peptide signal than trypsin digestion, and concentrating the protein prior to denaturation was necessary to achieve good signature peptide signal. The method had 5 pM LOD for BDNF from 8 uL digest and can be potentially applied for detecting these neurotrophins from in vivo sample.