On-Column Dimethylation with Capillary Liquid Chromatography-Tandem Mass Spectrometry for Online Determination of Neuropeptides in Rat Brain Microdialysate

Abstract

We have developed a method for online collection and quantitation of neuropeptides in rat brain microdialysates using on-column dimethylation with capillary liquid chromatography-tandem mass spectrometry (cLC-MS²). This method addresses a number of the challenges of quantifying neuropeptides with cLC-MS. It is also a completely automated and robust method for the preparation of stable isotope labeled-peptide internal standards to correct for matrix effects and thus ensure accurate quantitation. Originally developed for tissue-derived proteomics samples ( Raijmakers et al. Mol. Cell. Proteomics 2008 , 7 , 1755 - 1762 ), the efficacy of on-column dimethylation for native peptides in microdialysate has not been demonstrated until now. We have modified the process to make it more amenable to the time scale of microdialysis sampling and to reduce the accumulation of nonvolatile contaminants on the column and, thus, loss of sensitivity. By decreasing labeling time, we have a temporal resolution of 1 h from sample loading to elution and our peptide detection limits are in the low pM range for 5 μL injections of microdialysate. We have demonstrated the effectiveness of this method by quantifying basal and potassium stimulated concentrations of the neuropeptides leu-enkephalin and met-enkephalin in the rat hippocampus. To our knowledge, this is the first report of quantitation of these peptides in the hippocampus using MS.

Figure 1

Schematic for two-column labeling setup. Valve 1 is housed on the autosampler, Valve 2 is housed in the LC column oven, and Valve 3 is an external valve housed on the MS stage. Valve 2 is a 10-port valve but is depicted with 6-ports for clarity. White indicates a valve that is switching. Labeling is performed in the following way: (A) A 5-μL sample loop on Valve 2 is filled with microdialysate (MD) and upon switching, the sample is directed onto the pre-column (PC) by the loading pump (LP); (B) A series of five autosampler (AS) injections delivers reagents from vials (In sequential order: light labeling reagent, formic acid, 100 pM peptide aqueous standards, heavy labeling reagent, and formic acid) onto the PC, which react with peptides that have adsorbed onto the PC; and (C) Valve 3 switches, directing flow from the analytical pump (AP) through the PC, eluting peptides off the PC and onto the analytical column-mass spectrometer (LC-MS) by gradient elution.

Figure 2

Calibration curve for LE using the one-column setup. The relative area is defined as the area of the light peptide (in Ringer’s) divided by the area of the heavy peptide (100 pM LE in water). Error bars represent the SEM for n = 2 replicates.

Figure 3

Quantitation of LE (blue), ME (black), and yaGfl (100 pM, red) (A) based on the peak area of the light labeled peptide and (B) based on the relative area, in which the peak area of the light peptide (in Ringer’s) is divided by the peak area of the heavy peptide (100 pM in water). Black squares with blue centers indicate overlapping data points. Error bars represent the SEM of n = 2 replicates

Figure 4

Profile of LE (blue) and ME (black) levels during sampling of a single rat. The first point (t=0 hours) was collected 15 minutes after probe implantation. Shaded areas represent points collected during 10-minute potassium stimulation. The relative area is defined as the peak area of the light peptide divided by the peak area of the heavy peptide.

Figure 5

Peptide detection during potassium stimulation. A) Chromatograms and corresponding mass spectra at the point of elution of B) light LE, C) heavy LE, D) light ME, and E) heavy ME during 10-minute stimulation with 100 mM KCl Ringer’s. Light and heavy yaGfl spectra can be found in Figure S6.