What are we imaging? Software tools and experimental strategies for annotation and identification of small molecules in mass spectrometry imaging

Abstract

Mass spectrometry imaging (MSI) has become a widespread analytical technique to perform nonlabeled spatial molecular identification. The Achilles' heel of MSI is the annotation and identification of molecular species due to intrinsic limitations of the technique (lack of chromatographic separation and the difficulty to apply tandem MS). Successful strategies to perform annotation and identification combine extra analytical steps, like using orthogonal analytical techniques to identify compounds; with algorithms that integrate the spectral and spatial information. In this review, we discuss different experimental strategies and bioinformatics tools to annotate and identify compounds in MSI experiments. We target strategies and tools for small molecule applications, such as lipidomics and metabolomics. First, we explain how sample preparation and the acquisition process influences annotation and identification, from sample preservation to the use of orthogonal techniques. Then, we review twelve software tools for annotation and identification in MSI. Finally, we offer perspectives on two current needs of the MSI community: the adaptation of guidelines for communicating confidence levels in identifications; and the creation of a standard format to store and exchange annotations and identifications in MSI.

MALDI Mass Spectrometry Imaging: A Potential Game-Changer in a Modern Microbiology

“…The analysis range of such apparatuses fluctuates from 2 to 20 kDa, which is suitable for the identification of microorganisms based on their protein fingerprints. However, TOF analyzers display relatively poor resolution and mass accuracy for low masses when mass resolution increases as m / z increases [ 122 ]. Hence, certain small metabolites could not be accurately visualized and identified because peptide fragmentation is not possible with these mass spectrometers.…”

“…In contrast to the TOF analyzer, the orbitrap mass resolution decreases as m/z increases [ 123 ]. Hence, it has a very high mass resolution for low-weight compounds [ 122 ]. Nevertheless, despite the wide spectrum of commercial products offering different specifications (e.g., Orbitrap Exploris™ 120–240-480 or IQ-X, Fusion, Eclipse, Ascend tribrid MS from ThermoFisher Scientific), orbitrap mass spectrometers require the addition of a third-party (AP)-MALDI source, and they are pricier in comparison to already implemented MALDI-TOF devices in routine laboratories.…”

“…Sample preparation is an essential step in MSI, as proper handling can save both distribution, i.e., prevent delocalization of analytes, and the abundance of biomolecules, as well as guarantee an optimized spatial resolution, a high sensitivity, better annotation and identification of molecules [ 122 , 124 ]. Nevertheless, to our knowledge, few studies focused on the optimization of MALDI samples preparation for the imaging of microorganisms [ 59 , 71 , 97 ].…”

Metabolization and sequestration of plant specialized metabolites in insect herbivores: Current and emerging approaches

“…Directly mapping PSMs in a histological section allows distinction of the exact tissue and cells in which they are present. However, not all compounds can be detected by standard histological procedures (for a review on strategies for annotation and identification of small molecules see Baquer et al, 2022 ). Mass spectrometry imaging (MSI) provides simultaneous and spatially resolved analysis of molecular species in an untargeted fashion, and therefore, has been widely used to map the distribution of a broad variety of molecular compounds in tissues, organs, or whole organisms.…”

Metabolization and Sequestration of Plant Specialized Metabolites in Insect Herbivores: Current and Emerging Approaches

“…Directly mapping PSMs in a histological section allows distinction of the exact tissue and cells in which it is present. However, not all compounds can be detected by standard histological procedures (for a review on strategies for annotation and identification of small molecules see Baquer et al (2022)). Mass spectrometry imaging (MSI) provides simultaneous and spatially resolved analysis of molecular species in an untargeted fashion, and therefore, has been widely used to map the distribution of a broad variety of molecular compounds in tissues, organs, or whole organism.…”