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.
Hypoxia and Metastasis Team and ‡Cell Communications Team, Cancer Research U.K. Tumour Cell Signalling Unit, Division of Cancer Biology, The Institute of Cancer Research , London, United Kingdom.
Marie-Claude
MC
Djidja
Joan
J
Chang
Andreas
A
Hadjiprocopis
Fabian
F
Schmich
John
J
Sinclair
Martina
M
Mršnik
Erwin M
EM
Schoof
Holly E
HE
Barker
Rune
R
Linding
Claus
C
Jørgensen
Janine T
JT
Erler
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D005260:Female; D000860:Hypoxia / Q000378:metabolism*; D007150:Immunohistochemistry; D007553:Isotope Labeling / Q000379:methods; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010455:Peptides / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000596:Amino Acids; D010455:Peptides; D011506:Proteins