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

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 ??-bag cell peptide [1-9] (??BCP). 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 ??BCP. 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 ??BCP 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.close192

Microfluidic Device for the Selective Chemical Stimulation of Neurons and Characterization of Peptide Release with Mass Spectrometry

Neuropeptides are synthesized in and released from neurons and are involved in a wide range of physiological processes, including temperature homeostasis, learning, memory, and disease. When working with sparse neuronal. net-works, the ability to collect and characterize small sample volumes is important as neurons often release only a small proportion of their mass limited content Microfluidic systems are well suited for the study of neuropeptides. They offer the ability to control and manipulate the extracellular environment and small sample volumes, thereby reducing the dilution of peptides following release. We present an approach for the culture and stimulation of a neuronal network within a microfluidic device, subsequent collection of the released peptides, and their detection via mass spectrometry. The system employs microvalve-controlled stimulation channels to selectively stimulate a low-density neuronal culture, allowing us to determine the temporal onset of peptide release. Released peptides from the well-characterized, peptidergic bag cell neurons of Aplysia californica were collected and their temporal pattern of release was characterized with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We show a robust difference in the timing of release for chemical solutions containing elevated K+ (7 +/- 3 min), when compared to insulin (19 +/- 7 min) (p < 0.00001).close