Measuring D-amino acid-containing neuropeptides with capillary electrophoresis.
ABSTRACT Neuropeptides are heavily posttranslationally modified (PTM) gene products that are often characterized by a variety of mass spectrometric approaches. Recently, the occurrence of amino acids in the D-form has been documented in several neuropeptides. As this modification has no associated mass shift, this particular PTM is difficult to evaluate using mass spectrometry (MS) alone. Here we demonstrate several approaches using capillary electrophoresis (CE) with absorbance and laser-induced fluorescence (LIF) for the separation of native and derivatized molluscan peptides containing D-amino acids. The combination of peptide derivatization followed by CE/LIF is well suited for single cell measurements because of its ability to characterize the peptides in such small samples. In order to verify this approach, the D-Trp-containing peptide NdWFa (NH2-Asn-D-Trp-Phe-CONH2), present in individual neurons from the marine mollusk Aplysia californica, has been characterized. The mass spectra show that NdWFa and/or NWFa are present in specific neurons; CE/LIF analysis of these cells demonstrates that NdWFa is the dominant form of the peptide.
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ABSTRACT: 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).Analytical Chemistry 10/2012; 84(21-21):9446-9452. DOI:10.1021/ac302283u · 5.83 Impact Factor
Article: Chemical Analysis of Single CellsAnalytical Chemistry 06/2011; 83(12):4369-92. DOI:10.1021/ac2009838 · 5.83 Impact Factor
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ABSTRACT: Traditionally, the D-amino acid-containing peptide (DAACP) candidate can be discovered by observing the differences of biological activity and chromatographic retention time between the synthetic peptides and naturally occurring peptides. However, it is difficult to determine the exact position of D-amino acid in the DAACP candidates. Herein, we developed a novel site-specific strategy to rapidly and precisely localize D-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the D/L-peptide epimers were separated by on-line reversed-phase liquid chromatography and fragmented by collision induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from D/L-peptide epimers exhibit conformational differences, thus showing different mobility in IMS. The arrival time shift between the epimeric fragment ions was used as criteria to localize the D-amino acid substitution. The utility of this strategy was demonstrated by analysis of peptide epimers with different molecular sizes, [D-Trp]-Melanocyte stimulating hormone, [D-Ala]-Deltorphin, [D-Phe]-Achatin-I and their counterparts that contain all-L amino acids. Furthermore, the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated from the American lobster H. americanus and identified by integration of MS-based bottom-up and top-down sequencing approaches. The IMS data acquired using our novel site-specific strategy localized the site of isomerization of L- to D-Phe at the third residue of the CHHs from the N-terminus. Collectively, this study demonstrates a new method for discovery of DAACPs using IMS technique with the ability to localize D-amino acid residues.Analytical Chemistry 12/2013; 86(6). DOI:10.1021/ac4033824 · 5.83 Impact Factor