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Capillary electrophoresis coupled to MALDI mass spectrometry imaging with large volume sample stacking injection for improved coverage of C. borealis neuropeptidome.

ABSTRACT: Neuropeptides are important signaling molecules responsible for a wide range of functions within the nervous and neuroendocrine system. However, they are difficult to study due to numerous challenges, most notably their large degree of variability and low abundance in vivo. As a result, effective separation methods with sensitive detection capabilities are necessary for profiling neuropeptides in tissue samples, particularly those of simplified model organisms such as crustaceans. In order to address these challenges, this study utilized a capillary electrophoresis (CE)-matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) platform, building upon our previous design for improved neuropeptidomic coverage. The capillary was coated with polyethylenimine (PEI) to reduce peptide adsorption and reverse the electroosmotic flow, and large volume sample stacking (LVSS) was used to load and pre-concentrate 1 ?L of sample. The method demonstrated good reproducibility, with lower than 5% relative standard deviation for standards, and a limit of detection of approximately 100 pM for an allatostatin III peptide standard. The method was tested on brain and sinus gland (SG) tissue extracts and enabled detection of over 200 neuropeptides per run. When comparing the number detected in brain extracts in a direct spot, 60-second fractions, and 30-second fractions, the continuous trace collection afforded by the CE-MALDI-MSI platform yielded the largest number of detected neuropeptides. The method was compared to conventional LC-ESI-MS, and though the number of neuropeptides detected by LC-ESI-MS was slightly larger, the two methods were highly complementary, indicating the potential for the CE-MALDI-MSI method to uncover previously undetected neuropeptides in the crustacean nervous system. These results indicate the potential of CE-MALDI-MSI for routine use in neuropeptide research.

SUBMITTER: DeLaney K

PROVIDER: S-EPMC6917920 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Capillary electrophoresis coupled to MALDI mass spectrometry imaging with large volume sample stacking injection for improved coverage of C. borealis neuropeptidome.

DeLaney Kellen K Li Lingjun L

The Analyst 20191201 1

Neuropeptides are important signaling molecules responsible for a wide range of functions within the nervous and neuroendocrine system. However, they are difficult to study due to numerous challenges, most notably their large degree of variability and low abundance in vivo. As a result, effective separation methods with sensitive detection capabilities are necessary for profiling neuropeptides in tissue samples, particularly those of simplified model organisms such as crustaceans. In order to ad ...[more]

PMID: 31723949

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Project description:The ability to characterize chemical heterogeneity in biological structures is essential to understanding cellular-level function in both healthy and diseased states, but these variations remain difficult to assess using a single analytical technique. While mass spectrometry (MS) provides sufficient sensitivity to measure many analytes from volume-limited samples, each type of mass spectrometric analysis uncovers only a portion of the complete chemical profile of a single cell. Matrix-assisted laser desorption/ionization (MALDI) MS and capillary electrophoresis electrospray ionization (CE-ESI)-MS are complementary analytical platforms frequently utilized for single-cell analysis. Optically guided MALDI MS provides a high-throughput assessment of lipid and peptide content for large populations of cells, but is typically nonquantitative and fails to detect many low-mass metabolites because of MALDI matrix interferences. CE-ESI-MS allows quantitative measurements of cellular metabolites and increased analyte coverage, but has lower throughput because the electrophoretic separation is relatively slow. In this work, the figures of merit for each technique are combined via an off-line method that interfaces the two MS systems with a custom liquid microjunction surface sampling probe. The probe is mounted on an xyz translational stage, providing 90.6 ± 0.6% analyte removal efficiency with a spatial targeting accuracy of 42.8 ± 2.3 ?m. The analyte extraction footprint is an elliptical area with a major diameter of 422 ± 21 ?m and minor diameter of 335 ± 27 ?m. To validate the approach, single rat pancreatic islet cells were rapidly analyzed with optically guided MALDI MS to classify each cell into established cell types by their peptide content. After MALDI MS analysis, a majority of the analyte remains for follow-up measurements to extend the overall chemical coverage. Optically guided MALDI MS was used to identify individual pancreatic islet ? and ? cells, which were then targeted for liquid microjunction extraction. Extracts from single ? and ? cells were analyzed with CE-ESI-MS to obtain qualitative information on metabolites, including amino acids. Matching the molecular masses and relative migration times of the extracted analytes and related standards allowed identification of several amino acids. Interestingly, dopamine was consistently detected in both cell types. The results demonstrate the successful interface of optical microscopy-guided MALDI MS and CE-ESI-MS for sequential chemical profiling of individual, mammalian cells.

Dataset Information

Capillary electrophoresis coupled to MALDI mass spectrometry imaging with large volume sample stacking injection for improved coverage of C. borealis neuropeptidome.

Publications

Capillary electrophoresis coupled to MALDI mass spectrometry imaging with large volume sample stacking injection for improved coverage of C. borealis neuropeptidome.

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