Spatially‐resolved protein surface microsampling from tissue sections using liquid extraction surface analysis

Abstract

Tissue microenvironment characterization presents a challenge for a better understanding of the full complexity of a pathology. Unfortunately, making a precise "picture" of the disease needs an efficient microsampling method coupled to an accurate localization for performing region-dependent proteomics. Here, we present a method that enables rapid and reproducible extraction of proteins from a tissue section to analyze a specific region at a millimeter scale. The method used a liquid-microjunction extraction with conventional detergent solution for proteomics analysis. We successfully performed immunoblotting experiments and showed the possibility to retrieve and identify more than 1400 proteins from a 1-mm diameter spot size on tissue sections with a high degree of reproducibility both qualitatively and quantitatively. Moreover, the small size of the extracted region achieved by this sampling method allows the possibility to perform multiple extractions on different tissue section points. Ten points on a sagittal rat brain tissue section were analyzed and the measured proteins clearly distinguished the different parts of the brain, thus permitting precise functional mapping. We thus demonstrate that with this technology, it is possible to map the tissue microenvironment and gain an understanding of the molecular mechanisms at millimeter resolution.

Combined Mass Spectrometry Imaging and Top-down Microproteomics Reveals Evidence of a Hidden Proteome in Ovarian Cancer

“…These regions were analyzed by MALDI-MSI and discerned by spatial segmentation analysis (Alexandrov et al, 2011, Bonnel et al, 2011, Bruand et al, 2011), and the proteins were microextracted utilizing LMJ and PAM approaches (Franck et al, 2013, Quanico et al, 2014, Quanico et al, 2013, Wisztorski et al, 2016). A total of 237 gene products within the three regions were identified.…”

“…We first performed MS imaging of lipids in order to perform spatial segmentation analysis to identify regions of interest (ROIs), which were then subjected to liquid micro-junction (LMJ) (Quanico et al, 2013, Wisztorski et al, 2016) or parafilm-assisted manual microdissection (PAM) (Franck et al, 2013, Quanico et al, 2014) methods of microextraction. LMJ and PAM were followed by top-down proteomics for protein identification from necrotic/fibrotic tumor, tumor and benign (B) regions (technical triplicate).…”

Ambient ionisation mass spectrometry for in situ analysis of intact proteins

“…Similar bottom‐up approaches have been applied to DBS (see details above) and formalin‐fixed paraffin‐embedded ovarian cancer tissue . Wisztorski et al have applied a bottom‐up approach for spatially directed extraction of intact proteins from thin tissue sections of mouse brain, prior to digestion and analysis of the resulting peptides by LC‐MS/MS . Over 1400 proteins were identified from a 1‐mm pixel location.…”

“…Moreover, proteins may be present over a wide concentration range, with higher abundance proteins masking the presence of lower abundance proteins. One potential approach for addressing these challenges is to incorporate liquid chromatography; however, a considerable disadvantage of liquid‐phase separation techniques is the time cost. A typical protein or peptide HPLC analysis takes tens of minutes to an hour, making that approach incompatible with mass spectrometry imaging.…”

MALDI mass spectrometry imaging: A cutting‐edge tool for fundamental and clinical histopathology

“…Attempts are being proposed to combine MSI and solvent-based tissue surface sampling to access more informative proteomics data from microanatomies. Currently, those approaches are limited to the detection of drugs [169], lipids [170], and proteins from fr/fr tissues [171].…”