Strategy for SRM-based verification of biomarker candidates discovered by iTRAQ method in limited breast cancer tissue samples.
Since LC-MS-based quantitative proteomics has become increasingly applied to a wide range of biological applications over the past decade, numerous studies have performed relative and/or absolute abundance determinations across large sets of proteins. In this study, we discovered prognostic biomarker candidates from limited breast cancer tissue samples using discovery-through-verification strategy combining iTRAQ method followed by selected reaction monitoring/multiple reaction monitoring analysis (SRM/MRM). We identified and quantified 5122 proteins with high confidence in 18 patient tissue samples (pooled high-risk (n=9) or low-risk (n=9)). A total of 2480 proteins (48.4%) of them were annotated as membrane proteins, 16.1% were plasma membrane and 6.6% were extracellular space proteins by Gene Ontology analysis. Forty-nine proteins with >2-fold differences in two groups were chosen for further analysis and verified in 16 individual tissue samples (high-risk (n=9) or low-risk (n=7)) using SRM/MRM. Twenty-three proteins were differentially expressed among two groups of which MFAP4 and GP2 were further confirmed by Western blotting in 17 tissue samples (high-risk (n=9) or low-risk (n=8)) and Immunohistochemistry (IHC) in 24 tissue samples (high-risk (n=12) or low-risk (n=12)). These results indicate that the combination of iTRAQ and SRM/MRM proteomics will be a powerful tool for identification and verification of candidate protein biomarkers.
Laboratory of Proteome Research, National Institute of Biomedical Innovation, Ibaraki, Japan.
Satoshi
S
Muraoka
Hideaki
H
Kume
Shio
S
Watanabe
Jun
J
Adachi
Masayoshi
M
Kuwano
Misako
M
Sato
Naoko
N
Kawasaki
Yoshio
Y
Kodera
Makoto
M
Ishitobi
Hideo
H
Inaji
Yasuhide
Y
Miyamoto
Kikuya
K
Kato
Takeshi
T
Tomonaga
D000595:Amino Acid Sequence; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001943:Breast Neoplasms / Q000175:diagnosis / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D058851:GPI-Linked Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011379:Prognosis; D013194:Staining and Labeling; D018709:Statistics, Nonparametric; D053719:Tandem Mass Spectrometry
D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D016326:Extracellular Matrix Proteins; C489339:GP2 protein, human; D058851:GPI-Linked Proteins; D006023:Glycoproteins; C400752:MFAP4 protein, human; D010446:Peptide Fragments