Ultra-High Mass Resolution MALDI Imaging Mass Spectrometry of Proteins and Metabolites in a Mouse Model of Glioblastoma

M Dilillo,M Caleo,E L De Graaf,M Costa,C Esteve,D Pellegrini,E Vannini,S Nicolardi,R Ait-Belkacem,L A Mcdonnell

doi:10.1038/s41598-017-00703-w

Abstract

MALDI mass spectrometry imaging is able to simultaneously determine the spatial distribution of hundreds of molecules directly from tissue sections, without labeling and without prior knowledge. Ultra-high mass resolution measurements based on Fourier-transform mass spectrometry have been utilized to resolve isobaric lipids, metabolites and tryptic peptides. Here we demonstrate the potential of 15T MALDI-FTICR MSI for molecular pathology in a mouse model of high-grade glioma. The high mass accuracy and resolving power of high field FTICR MSI enabled tumor specific proteoforms, and tumor-specific proteins with overlapping and isobaric isotopic distributions to be clearly resolved. The protein ions detected by MALDI MSI were assigned to proteins identified by region-specific microproteomics (0.8 mm2 regions isolated using laser capture microdissection) on the basis of exact mass and isotopic distribution. These label free quantitative experiments also confirmed the protein expression changes observed by MALDI MSI and revealed changes in key metabolic proteins, which were supported by in-situ metabolite MALDI MSI.

Highlights

To reduce the potential for false positives the 15 T MALDI-FTICR MSI results were aligned with the protein identifications obtained from LC-MS/MS analysis of extracts of small (0.8 mm2) defined histological areas isolated by laser capture microdissection (LCM)
The ultra-high mass resolution of T FTICR MSI allowed the detection of intact proteins between 3.5 and kDa with full isotopic resolution, it led to the detection of a much larger number of distinct protein ions, distinguished proteoforms and enabled the charge states and adduct types of many protein ions to be determined
15 T MALDI-FTICR MSI was used for ultra-high mass resolution, accurate mass MSI analysis of proteins and metabolites in a murine model of glioblastoma

Summary

Introduction

Overlapping, non-resolved mass spectral peaks undermine the ability to identify biomarkers because disease associated changes in expression are diluted by overlapping peaks; an effect similar to the ratio compression detected in LC-MS/MS with isobaric labeling strategies due to interfering co-eluting peptides[12, 13]. We report an ultra-high mass resolution study, performed using a 15 T MALDI-FTICR mass spectrometer, of a murine model of glioblastoma multiforme (GBM). The ultra-high mass resolution enabled GBM-associated proteoforms to be resolved, including proteins with interspersed and isobaric isotopomers. The presence of isobaric, isotopomer protein ions complicates the assignment of their identity, by comparison with LC-MS/MS analysis of protein extracts, because of the increased potential for false positives. To reduce the potential for false positives the 15 T MALDI-FTICR MSI results were aligned with the protein identifications obtained from LC-MS/MS analysis of extracts of small (0.8 mm2) defined histological areas isolated by laser capture microdissection (LCM)

Methods

Results

Conclusion