Proteome profiling of breast tumors by gel electrophoresis and nanoscale electrospray ionization mass spectrometry.
We have conducted proteome-wide analysis of fresh surgery specimens derived from breast cancer patients, using an approach that integrates size-based intact protein fractionation, nanoscale liquid separation of peptides, electrospray ion trap mass spectrometry, and bioinformatics. Through this approach, we have acquired a large amount of peptide fragmentation spectra from size-resolved fractions of the proteomes of several breast tumors, tissue peripheral to the tumor, and samples from patients undergoing noncancer surgery. Label-free quantitation was used to generate protein abundance maps for each proteome and perform comparative analyses. The mass spectrometry data revealed distinct qualitative and quantitative patterns distinguishing the tumors from healthy tissue as well as differences between metastatic and non-metastatic human breast cancers including many established and potential novel candidate protein biomarkers. Selected proteins were evaluated by Western blotting using tumors grouped according to histological grade, size, and receptor expression but differing in nodal status. Immunohistochemical analysis of a wide panel of breast tumors was conducted to assess expression in different types of breast cancers and the cellular distribution of the candidate proteins. These experiments provided further insights and an independent validation of the data obtained by mass spectrometry and revealed the potential of this approach for establishing multimodal markers for early metastasis, therapy outcomes, prognosis, and diagnosis in the future.
Alldridge
Louise
L
The Helen Rollason Research Laboratory, Helen Rollason Heal Cancer Charity, Chelmsford, Essex CM1 1LL, United Kingdom.
Metodieva
Gergana
G
Greenwood
Christina
C
Al-Janabi
Khalid
K
Thwaites
Laura
L
Sauven
Paul
P
Metodiev
Metodi
M
D014408:Biomarkers, Tumor / Q000032:analysis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D054402:Chemokine CCL1 / Q000032:analysis; D002853:Chromatography, Liquid / Q000379:methods; D016000:Cluster Analysis; D019295:Computational Biology; D004268:DNA-Binding Proteins / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D018398:Homeodomain Proteins / Q000032:analysis; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis; D008840:Microfilament Proteins / Q000032:analysis; D046210:Microfluidic Analytical Techniques; D000071317:Nanog Homeobox Protein; D036103:Nanotechnology; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D050794:STAT1 Transcription Factor / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013939:Thymidine Phosphorylase / Q000032:analysis; D015854:Up-Regulation
D014408:Biomarkers, Tumor; C065971:CCL1 protein, human; D054402:Chemokine CCL1; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C047262:LCP1 protein, human; D008565:Membrane Proteins; D008840:Microfilament Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D020543:Proteome; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D013939:Thymidine Phosphorylase