Label-free quantitation of peptide release from neurons in a microfluidic device with mass spectrometry imaging

Abstract

Microfluidic technology allows the manipulation of mass-limited samples and when used with cultured cells, enables control of the extracellular microenvironment, making it well suited for studying neurons and their response to environmental perturbations. While matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) provides for off-line coupling to microfluidic devices for characterizing small-volume extracellular releasates, performing quantitative studies with MALDI is challenging. Here we describe a label-free absolute quantitation approach for microfluidic devices. We optimize device fabrication to prevent analyte losses before measurement and then incorporate a substrate that collects the analytes as they flow through a collection channel. Following collection, the channel is interrogated using MS imaging. Rather than quantifying the sample present via MS peak height, the length of the channel containing appreciable analyte signal is used as a measure of analyte amount. A linear relationship between peptide amount and band length is suggested by modeling the adsorption process and this relationship is validated using two neuropeptides, acidic peptide (AP) and α-bag cell peptide [1-9] (αBCP). The variance of length measurement, defined as the ratio of standard error to mean value, is as low as 3% between devices. The limit of detection (LOD) of our system is 600 fmol for AP and 400 fmol for αBCP. Using appropriate calibrations, we determined that an individual Aplysia bag cell neuron secretes 0.15 ± 0.03 pmol of AP and 0.13 ± 0.06 pmol of αBCP after being stimulated with elevated KCl. This quantitation approach is robust, does not require labeling, and is well suited for miniaturized off-line characterization from microfluidic devices.

Microfluidics-to-mass spectrometry: A review of coupling methods and applications

“…The Sweedler group demonstrated a method for quantitation of analyte concentration using MALDI to image adsorption of the analyte along a length of a PDMS microfluidic channel [66]. They demonstrated through simulations and experiments that the length of the channel containing analyte was linearly proportional to the concentration of analyte (Figure 22A).…”

“…(C) Calibration curve for relating the channel coverage length to the concentration of analyte. Adapted with permission from [66]. Copyright (2012) Royal Society of Chemistry.…”

Mass spectrometry-based characterization of endogenous peptides and metabolites in small volume samples

“…Quantitative analyses of released analytes can also be performed using a microfluidic platform hyphenated to MS. Zhong et al [102] developed a device for the quantification of peptides released from A. californica bag cell neurons. The device consisted of one serpentine channel treated with octadecyltrichlorosilane (OTS), used to collect the peptides at specific locations.…”

Small-Volume Analysis of Cell–Cell Signaling Molecules in the Brain

“…A number of lab-on-a-chip applications have focused efforts toward improving signaling molecule detection, including sample preparation on chip (Wei et al, 2010), surface modifications (Jo et al, 2007;Zhong et al, 2012), and on-chip detection (Dishinger et al, 2009). The Kennedy group (Dishinger et al, 2009) reported an automated approach that quantified insulin release from 15 single islet cells using an on-chip electrophoresis channel and an immune assay for detection.…”

“…The Kennedy group (Dishinger et al, 2009) reported an automated approach that quantified insulin release from 15 single islet cells using an on-chip electrophoresis channel and an immune assay for detection. We reported a microfluidic system with C18 functionalized channels to capture and quantify neuropeptide release on-chip with MS detection (Zhong et al, 2012). Rather than quantifying the peptide level via MS peak height, the length of the channel producing appreciable peptide signal from adsorption was used as a measure of peptide amount in release.…”