MicroLESA: Integrating Autofluorescence Microscopy, In Situ Micro-Digestions, and Liquid Extraction Surface Analysis for High Spatial Resolution Targeted Proteomic Studies

Ryan, Daniel; Patterson, Nathan Heath; Putnam, Nicole E.; Wilde, Aimee D; Weiss, Amy L.; Perry, William; Cassat, James E.; Skaar, Eric P.; Caprioli, Richard M.; Spraggins, Jeffrey M.

doi:10.1021/acs.analchem.8b05889

Cited by 56 publications

(59 citation statements)

References 90 publications

(158 reference statements)

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“…Previous in vitro studies have focused on the metabolic capabilities of S. aureus in the absence of glucose, as the centers of abscesses are predicted to be glucose-limited (21). Transcript and protein levels of gluconeogenic enzymes in S. aureus have also been found to be elevated during invasive infection, and in OM specifically (22,23). Our studies, however, suggest that S. aureus requires glycolysis and glycolytic carbon sources to survive in vivo during OM.…”

Section: Discussionmentioning

confidence: 58%

Host nutrient milieu drives an essential role for aspartate biosynthesis during invasive Staphylococcus aureus infection

Potter

Butrico

Ford

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The bacterial pathogenStaphylococcus aureusis capable of infecting a broad spectrum of host tissues, in part due to flexibility of metabolic programs.S. aureus, like all organisms, requires essential biosynthetic intermediates to synthesize macromolecules. We therefore sought to determine the metabolic pathways contributing to synthesis of essential precursors during invasiveS. aureusinfection. We focused specifically on staphylococcal infection of bone, one of the most common sites of invasiveS. aureusinfection and a unique environment characterized by dynamic substrate accessibility, infection-induced hypoxia, and a metabolic profile skewed toward aerobic glycolysis. Using a murine model of osteomyelitis, we examined survival ofS. aureusmutants deficient in central metabolic pathways, including glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and amino acid synthesis/catabolism. Despite the high glycolytic demand of skeletal cells, we discovered thatS. aureusrequires glycolysis for survival in bone. Furthermore, the TCA cycle is dispensable for survival during osteomyelitis, andS. aureusinstead has a critical need for anaplerosis. Bacterial synthesis of aspartate in particular is absolutely essential for staphylococcal survival in bone, despite the presence of an aspartate transporter, which we identified as GltT and confirmed biochemically. This dependence on endogenous aspartate synthesis derives from the presence of excess glutamate in infected tissue, which inhibits aspartate acquisition byS. aureus. Together, these data elucidate the metabolic pathways required for staphylococcal infection within bone and demonstrate that the host nutrient milieu can determine essentiality of bacterial nutrient biosynthesis pathways despite the presence of dedicated transporters.

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Section: Discussionmentioning

confidence: 58%

Host nutrient milieu drives an essential role for aspartate biosynthesis during invasive Staphylococcus aureus infection

Potter

Butrico

Ford

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Label-free autofluorescence microscopy for functional and morphological tissue analyses is currently experiencing a true renaissance in life sciences, also driven by the rapidly developing techniques for image registration and mapping 22 and the capabilities of deep machine learning in detection, recognition and analysis of histo(morpho)logical patterns 48 . For correlation of MSI data with histomorphology, previous studies have already demonstrated the beneficial combination of autofluorescence microscopy with MALDI-MSI in multimodal analyses of (2D)-tissue sections 21 , 22 . Here, we used autofluorescence- and label-based LSFM, to define different regions of interest (ROIs) in optically cleared, 3-dimensional brain tissue samples for subsequent examination by MALDI-MSI.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, MALDI-MSI analyses can also be combined with a variety of other imaging modalities, providing additional morphological and/or biochemical information from the identical tissue section, or sample 14 , 17 . Previously reported multimodal (MALDI)-MSI analysis approaches e.g., include the combination with autofluorescence-microscopy 21 , 22 , Raman spectroscopy 23 , magnetic resonance imaging (MRI) 24 , 25 , histology, immunohistochemistry, and laser scanning confocal microscopy 26 . Using appropriate alignment methods 22 , 24 , 27 , the images acquired by MALDI-MSI and additional imaging modalities can be exactly overlaid and thus directly be related to e.g., distinct morphological features of the examined tissue, such as different structural compartments, cell types, pathological alterations, etc.…”

Section: Introductionmentioning

confidence: 99%

Light sheet fluorescence microscopy guided MALDI-imaging mass spectrometry of cleared tissue samples

Blutke

Sun

et al. 2020

Sci Rep

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Light sheet fluorescence microscopy (LSFM) of optically cleared biological samples represents a powerful tool to analyze the 3-dimensional morphology of tissues and organs. Multimodal combinations of LSFM with additional analyses of the identical sample help to limit the consumption of restricted specimen and reduce inter-sample variation. Here, we demonstrate the proof-of-concept that LSFM of cleared brain tissue samples can be combined with Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) for detection and quantification of proteins. Samples of freshly dissected murine brain and of archived formalin-fixed paraffin-embedded (FFPE) human brain tissue were cleared (3DISCO). Tissue regions of interest were defined by LSFM and excised, (re)-embedded in paraffin, and sectioned. Mouse sections were coated with sinapinic acid matrix. Human brain sections were pre-digested with trypsin and coated with α-cyano-4-hydroxycinnamic acid matrix. Subsequently, sections were subjected to MALDI-time-of-flight (TOF)-MSI in mass ranges between 0.8 to 4 kDa (human tissue sections), or 2.5–25 kDa (mouse tissue sections) with a lateral resolution of 50 µm. Protein- and peptide-identities corresponding to acquired MALDI-MSI spectra were confirmed by parallel liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis. The spatial abundance- and intensity-patterns of established marker proteins detected by MALDI-MSI were also confirmed by immunohistochemistry.

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“…Direct coupling to high-performance liquid chromatography for analyte separation and concentration prior to mass analysis is also possible and can be performed with commercial systems [18,21]. Recent bottom-up and top-down proteomics studies have been performed in this way and provide complementary data to conventional MSI experiments, with higher spatial resolution (∼110 μm) recently achieved with the microLESA setup [22][23][24]. Since the spatial resolution is largely governed by the available compatible pipette tips, modified systems demonstrating LESA at smaller sampling areas may be developed in the future.…”

Section: Liquid Extraction Surface Analysis (Lesa)mentioning

confidence: 99%

In situ mass spectrometry analysis of intact proteins and protein complexes from biological substrates

Hale

Cooper

2020

Biochemical Society Transactions

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Advances in sample preparation, ion sources and mass spectrometer technology have enabled the detection and characterisation of intact proteins. The challenges associated include an appropriately soft ionisation event, efficient transmission and detection of the often delicate macromolecules. Ambient ion sources, in particular, offer a wealth of strategies for analysis of proteins from solution environments, and directly from biological substrates. The last two decades have seen rapid development in this area. Innovations include liquid extraction surface analysis, desorption electrospray ionisation and nanospray desorption electrospray ionisation. Similarly, developments in native mass spectrometry allow protein–protein and protein–ligand complexes to be ionised and analysed. Identification and characterisation of these large ions involves a suite of hyphenated mass spectrometry techniques, often including the coupling of ion mobility spectrometry and fragmentation techniques. The latter include collision, electron and photon-induced methods, each with their own characteristics and benefits for intact protein identification. In this review, recent developments for in situ protein analysis are explored, with a focus on ion sources and tandem mass spectrometry techniques used for identification.

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MicroLESA: Integrating Autofluorescence Microscopy, In Situ Micro-Digestions, and Liquid Extraction Surface Analysis for High Spatial Resolution Targeted Proteomic Studies

Abstract: The ability to target discrete features within tissue using liquid surface extractions enables the identification of proteins while maintaining the spatial integrity of the sample. Here, we present a *

Cited by 56 publications

References 90 publications

Host nutrient milieu drives an essential role for aspartate biosynthesis during invasive Staphylococcus aureus infection

Host nutrient milieu drives an essential role for aspartate biosynthesis during invasive Staphylococcus aureus infection

Light sheet fluorescence microscopy guided MALDI-imaging mass spectrometry of cleared tissue samples

In situ mass spectrometry analysis of intact proteins and protein complexes from biological substrates

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