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

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.