High Throughput In Situ DDA Analysis of Neuropeptides by Coupling Novel Multiplex Mass Spectrometric Imaging (MSI) with Gas-Phase Fractionation

Abstract

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) is a powerful tool to map the spatial distribution of biomolecules on tissue sections. Recent developments of hybrid MS instruments allow combination of different types of data acquisition by various mass analyzers into a single MSI analysis, which reduces experimental time and sample consumptions. Here, using the well-characterized crustacean nervous system as a test-bed, we explore the utility of high resolution and accurate mass (HRAM) MALDI Orbitrap platform for enhanced in situ characterization of the neuropeptidome with improved chemical information. Specifically, we report on a multiplex-MSI method, which combines HRAM MSI with data dependent acquisition (DDA) tandem MS analysis in a single experiment. This method enables simultaneous mapping of neuropeptide distribution, sequence validation and novel neuropeptide discovery in crustacean neuronal tissues. To enhance the dynamic range and efficiency of in situ DDA, we introduced a novel approach of fractionating full m/z range into several sub-mass ranges and embedding the setup using the multiplex-DDA-MSI scan events to generate pseudo fractionation before MS/MS scans. The division of entire m/z into multiple segments of m/z subranges for MS interrogation greatly decreased the complexity of molecular species from tissue samples and the heterogeneity of the distribution and variation of intensities of m/z peaks. By carefully optimizing the experimental conditions such as the dynamic exclusion, the multiplex-DDA-MSI approach demonstrates better performance with broader precursor coverage, less biased MS/MS scans towards high abundance molecules and improved quality of tandem mass spectra for low intensity molecular species.

Characterizing and alleviating ion suppression effects in atmospheric pressure matrix‐assisted laser desorption/ionization

“…8–9 Of note, AP-MALDI MSI enables sampling at semi-physiological conditions (i.e., at physiological temperature and pressure), which minimizes vacuum-induced sample alteration and the loss of volatile analytes. 10 Compared to vacuum MALDI sources hyphenated with the Orbitrap XL platform equipped with a nitrogen laser (60 Hz, 337 nm), 11–12 one of the distinct merits of the AP-MALDI-Orbitrap is the feasibility of achieving higher spatial resolution during tissue imaging analysis. Furthermore, the decreased laser spot size in AP MALDI source, together with collisional cooling under AP condition than that of under vacuum condition, helps to alleviate in-plume thermal and photochemical fragmentation, which might contribute to the gentle/soft nature of AP MALDI and better reproducibility compared to vacuum MALDI.…”

“…2A shows the dependency trend of intensity ratios of insulin to FMRF and Aβ(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) to FMRF as a function of laser energy. The lower intensity ratio of higher mass analytes to lower mass analytes indicates higher ISE during ionization.…”

Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights

“…By implementing a spiral plate motion during imaging, the integrity of spatial information was not lost with this method. 27 …”

Understanding interactions between biomaterials and biological systems using proteomics

“…As a result, the complexity of molecular species from tissue samples and the heterogeneity of the distribution and variation of intensities of m/z peaks are greatly decreased. By carefully optimising the experimental conditions such as the dynamic exclusion, the multiplex-DDA-MSI approach demonstrates better performance with broader precursor coverage, less biased MS/MS scans towards high abundance molecules and improved quality of tandem mass spectra for low intensity molecular species [148].…”