Analytical Chemistry Article Figure 2. Series of representative mass spectra of bag cell neuron peptide release following KCl stimulation at increasing stimulation exposure times. A number of previously identiﬁed peptides from the bag cell neurons were observed following 5 min of exposure to KCl (βBCP, m/z 728.4; αBCP1−7, m/z 922.5; αBCP, m/z 1122.6; AP, m/z 2959.5; pELH30−43, m/z 1471.8; δBCP1−36, m/z 4022.7; δBCP, m/z 4406.9). On the right of the mass spectra are an example of a time course for the chemical stimulation protocol series. Red bars represent chemical stimulation addition; gray bars indicate ASW addition. Each exposure was 40 min in duration with stimulation time and ASW addition varying. Two collections were done during each exposure time at t = 20 min and t = 40 min. via ﬂow-induced stress and motions across the culture chamber or by damaging the cells upon chemical stimulation. Each neural network was exposed to elevated K+ for a combination of exposure times. For example, a group of neurons may have been exposed to K+ for 1 min, 3 min, 5 min, 10 min, and 20 min or a subset of these exposure times, sequentially, with ASW as the pre- and postcontrol. Following each stimulation exposure, the microvalves were closed and ASW was ﬂowed through the device until the total time reached 40 min, allowing any released peptides to be washed out of the device for collection. Solutions were collected twice during the 40 min period to gain additional temporal release information. After collection, samples were desalted and concentrated with C18 pipet tips and eluted onto a MALDI target for analysis. In this study, nine diﬀerent groups of bag cell neurons, each in separate devices, received increasing periods of K+ stimulations and peptide release was detected. The resulting mass spectra were collected twice during each exposure time. Figure 2 shows a representative series of intensity-normalized mass spectra following 1, 3, 5, and 10 min exposures to K+. A number of well-studied peptides were detected following 5 min of K+ exposure, demonstrating that a 5 min exposure is suﬃcient to cause release. In general, peptide release was observed in both 20 min collections after the 5 min stimulation period (n = 4; out of seven devices where peptide release was observed). Peptides were not observed in the pre- and postconditions, indicating that the shear stress caused by ﬂow does not stimulate or lyse the cells. Because of its relatively good detection limits compared with the other egg-laying hormone-related peptides, the detection of acidic peptide (m/z 2959.5) at S/N ≥ 5 was used to indicate when peptide release had occurred. Figure 3 shows the percentage of the time that acidic peptide was detected versus the elevated K+ exposure time. Figure 3. Percentage of the time that acidic peptide (m/z 2959.5), a known peptide of the bag cell neurons from Aplysia californica, is detected versus stimulation exposure time for KCl and insulin. The n represents the number of distinct repeats (individual devices containing a distinct network of neurons) used that were stimulated for the indicated time. Stimulation time courses were varied to demonstrate that peptide release is independent of previously applied stimulations to the cells. (Top) 5 min of KCl exposure time was necessary to elicit peptide release 79% of the time. (Bottom) 20 min of insulin exposure was necessary to elicit peptide release 88% of the time. 9449 dx.doi.org/10.1021/ac302283u | Anal. Chem. 2012, 84, 9446−9452