Identification of Hypoxia-Regulated Proteins Using MALDI-Mass Spectrometry Imaging Combined with Quantitative Proteomics

Abstract

Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed.

Cancer cell adaptation to hypoxia involves a HIF‐GPRC5A‐YAP axis

“…Regions of hypoxia are frequently found in colorectal cancers (Yoshimura et al, 2004;Dekervel et al, 2014), but previous omics studies of hypoxia-mediated cellular responses have largely focused on breast cancer cells (Mole et al, 2009;Djidja et al, 2014;Semenza, 2017). To identify novel hypoxia-induced proteins in colorectal cancer cells, we performed SILAC-based proteomics in SW620 cells grown in normoxia (21% O 2 ) or hypoxia (1% O 2 ).…”

The role of hypoxia in inflammatory disease (Review)

“…Proteomics approaches have been used to identify protein changes in response to hypoxia in comparison with gene changes. Changes in just over 100 proteins in response to hypoxia have been identified (37,38). However, proteins identified represent both known and undescribed HIF targets, raising the possibility of HIF action outside of the conventional canonical pathway.…”

A MALDI-MSI Approach to the Characterization of Radiation-Induced Lung Injury and Medical Countermeasure Development

“…This molecular imaging technique has been applied to the investigation of lipids, metabolites and proteins in a plethora of pathophysiological processes(Carter et al 2011, Palmer et al 2012, Steven and Bunch 2013, Berry et al 2014, Djidja et al 2014, Jackson et al 2014, Jones et al 2014, Manier et al 2014, Seeley et al 2014, Shariatgorji et al 2014). By applying mass spectrometry to region-specific anatomical tissue architecture, MALDI-MSI offers another dimension in characterizing the natural history and development of these pathologies.…”