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Mass Spectrometry Imaging, Microdissection and LC-MS/MS of the Same Tissue Section Marialaura Dilillo, Davide Pellegrini, Rima Ait-Belkacem, Erik L. de Graaf, Matteo Caleo, and Liam A. McDonnell J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00284 • Publication Date (Web): 25 Jun 2017 Downloaded from http://pubs.acs.org on June 29, 2017

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Journal of Proteome Research

Mass Spectrometry Imaging, Laser Capture Microdissection and LC-MS/MS of the Same Tissue Section

Marialaura Dilillo1,2, Davide Pellegrini1,3, Rima Ait-Belkacem1, Erik L. de Graaf1, Matteo Caleo4, Liam A. McDonnell1,5* 1

Fondazione Pisana per la Scienza ONLUS, Pisa, Italy

2

Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa, Italy.

3

NEST, Scuola Normale Superiore di Pisa, Pisa, Italy

4

CNR Neuroscience Institute, Pisa, Italy

5

Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The

Netherlands

* Corresponding author and reprint requests: email: [email protected]

1 ACS Paragon Plus Environment


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Abstract Mass spectrometry imaging (MSI) is able to simultaneously record the distributions of hundreds of molecules directly from tissue. Rapid direct tissue analysis is essential for MSI, in order to maintain spatial localization and acceptable measurement times. The absence of an explicit analyte separation/purification step means MSI lacks the depth-of-coverage of LC-MS/MS. In this work we demonstrate how atmospheric pressure MALDI-MSI enables the same tissue section to be first analyzed by MSI, to identify regions-of-interest that exhibit distinct molecular signatures, followed by localized proteomics analysis using laser capture microdissection isolation and LC-MS/MS.

Keywords: Mass spectrometry imaging, microproteomics, laser capture microdissection, molecular histology, localized microproteomics, multimodal analysis.


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Introduction Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is able to simultaneously record the distributions of hundreds of molecules directly from tissue, without prior knowledge and without labeling1. MALDI MSI is routinely aligned with histology so that molecular signatures can be extracted from distinct organs or regions of tissue. In clinical research these capabilities are exploited for the identification of biomarkers to aid patient diagnosis, prognosis, and even predict response to therapy; in pharmacological research the combination of MALDI MSI and histology is used to assess tissue levels and distributions of drugs and their metabolites2–8. The direct-tissue-analysis nature of MALDI MSI is essential to maintain the original spatial distribution of the tissue’s molecular content, as well as for the high data acquisition speeds necessary for practical experiment times (especially so given the recent developments toward higher spatial resolution analysis). The direct-tissue-analysis nature of MALDI MSI coupled with the very small number of cells that contribute to each pixel’s mass spectrum (a 100×100µm pixel of a 10µm thick tissue section contains 20X increase in the number of identified peptides, >20X increase in the number of identified proteins and protein groups. The combination of MSI with high performance microproteomics, here using label-free analysis of on-tissue digested samples and the SP3 method, is applicable to all future developments that further improve the sensitivity/depth-of-coverage of LC-MS/MS experiments. Recent reports have demonstrated how the AssayMAP Bravo automated liquid handling platform can be configured for robust and ultrasensitive phosphoproteomics and high precision relative quantitation based on multiplex isobaric labeling32,37. In the latter example 1 mm2 and 0.5 mm2 areas of tissue were microdissected from 15 µm thick kidney tissue sections (note 10 µm thick tissue sections used here) then processed using the SP3 method and on-column desalting, TMT labeling, and high pH fractionation. These 10-plex TMT isobaric labeling experiments identified



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5002 and 3440 protein groups from the 1 mm2 and 0.5 mm2 areas respectively, >90% of which were also quantified. The phosphoproteomics experiment used more cells, 200 000, but when projected onto tissue sections the increased sensitivity provides capabilities that approach the length scales of the ROI’s typically highlighted by MSI: assuming an average cell size of 10 µm and tissue sections 10 µm thick, 200 000 cells corresponds to a 4 mm x 5 mm sized region of tissue, thus indicating the potential for MSI-defined or histology-defined phosphoproteomics. The combination of MSI with high performance microproteomics is clearly applicable to many biomedical applications, for example to gain more insight into regions highlighted by MSI as phenotypic tumor subpopulations3, as regions were drugs cross the blood brain barrier38, or regions with distinct metabolic behavior39. It could also be applied to further improve the MSI method itself. A number of on-tissue digestion methodology investigations have been reported35,40–44. The method used here was chosen because it was previously shown to provide more complete proteolysis, lower sensitivity to the tissue’s histology, and the greatest reproducibility35,36. However to date no report has explicitly determined the proportion of the available amount of each protein that is effectively sampled by MALDI MSI. The ability to perform AP-MALDI, LCM and LC-MS/MS of the same tissue section, if combined with isotopically labeled reference peptides for absolute quantitation of both MALDI and LC-MS/MS datasets, would enable the proportion of peptides that are sampled by MALDI MSI to be determined, precisely because they are from the same localized regions of tissue. Finally Rizzo et al. recently reported a hydrogel based approach for spatially localized protein extraction and digestion prior to LC-MS/MS analysis, and demonstrated that hundreds of proteins could be identified with hydrogel diameters of just 260 µm45. This hydrogel approach represents an elegant alternative for localized proteomics but further work is necessary to


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ascertain its compatibility with tissue sections previously prepared for and analyzed by MALDI MSI.

Concluding remarks With suitable reference standards MSI has been used to quantify the amounts and distributions of specific target molecules. However proteome/lipidome/metabolome-wide quantitation remains an unfulfilled challenge. The very small number of cells in each pixel, and the absence of any explicit separation technique, limits MALDI MSI to the more abundant species present in the tissue. It is for these reasons that MALDI MSI is most suited as a molecular histology technique that characterizes tissues on the basis of mass spectral signatures (so-called molecular histology). Here we demonstrated that AP-MALDI-MSI, LCM and LC-MS/MS may be performed on the same tissue section with no information loss. Thus MSI may be used to investigate the molecular histology of tissues, LCM then used to isolate specific regions characterized by distinct mass spectral profiles, and LC-MS/MS used to characterize those regions with the depth of coverage and quantitative ability of modern day biomolecular mass spectrometry (while avoiding the inevitable variability that is introduced when adjacent sections are used for MSI and LCMS/MS).

Supporting Information The following files are available free of charge at ACS website http://pubs.acs.org: ProteinIDs_SP3cortex.xlsx Protein identification information from triplicate analysis of microdissected, 1mm2 area, 10 µm thick, regions of mouse brain cortex.



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Acknowledgements This study was supported in part by AIRC (grant IG18925, M.C.) and the ERA-NET Transcan II project ARREST (number 166, LMD).

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Figure 1. Comparison AP-MALDI MS spectra of BSA digest from glass vs. PEN-coated slides. The total signal intensity for both mass spectra is approximately 1e2. BSA tryptic peptides denoted with asterisks. Note the lower background and absence of interfering peaks from the polymeric coating in the mass spectrum from the PEN coated slide.



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Figure 2. Experimental workflow for assessing the impact of AP-MALDI-MSI on quantitative LCM-MS/MS of microdissected regions of tissue. 1) Consecutive tissue sections were each mounted onto PEN coated slides and subject to on-tissue digestion using trypsin. 2) One set of tissue sections (top row) were then coated with α-CHCA matrix and analyzed by AP-MALDI MSI. 3) Regions of interest were then defined on the basis of the MSI data, and projected on to the control set of tissue sections, not analyzed by AP-MALDI MSI (bottom row). 4) The ROIs were isolated from the MSI and non-MSI tissue sections and compared using AP-MALDI mass spectral profiling and quantitative LC-MS/MS.


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Figure 3. Comparison of the AP-MALDI mass spectra of extracts from the MSI analyzed and control microdissected tissue samples. Selection of ROIs for LCM was based on the intensities of the peptide ions detected at m/z 1131.57 (TTHYGSLPQK from myelin basic protein

10

) and

m/z 1025.50 (ADLAEEYSK from Ubiquitin conjugating enzyme10), from which small 1.0 mm2 regions were isolated and are indicated with colored squares.



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Figure 4. Numbers of proteins and peptides identified by LC-MS/MS. A) and B) compare the numbers of protein groups and peptide groups, respectively, identified from microdissected locations of the MSI-analyzed, on-tissue digested tissue section and the control (not prepared for MSI or analyzed by MSI), on-tissue digested tissue section. C) Summary of the LC-MS/MS results from the mouse cortex microdissected tissue using on-tissue digestion (MSI analyzed and control tissues). D) Summary of the LC-MS/MS results from the mouse cortex microdissected tissue using SP3 method. * indicates p < 0.05 using a F-test and a t-test.

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