Quantitative profiling of drug-associated proteomic alterations by combined 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling and 2DE/MS identification.
The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.
Ou
Keli
K
Agenica Research/National Cancer Centre/Genome Institute of Singapore, 11 Hospital Drive, Singapore 169610.
Kesuma
Djohan
D
Ganesan
Kumaresan
K
Yu
Kun
K
Soon
Sou Yen
SY
Lee
Suet Ying
SY
Goh
Xin Pei
XP
Hooi
Michelle
M
Chen
Wei
W
Jikuya
Hiroyuki
H
Ichikawa
Tetsuo
T
Kuyama
Hiroki
H
Matsuo
Ei-ichi
E
Nishimura
Osamu
O
Tan
Patrick
P
D045744:Cell Line, Tumor; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D006801:Humans; D015151:Immunoblotting; D007553:Isotope Labeling; D013058:Mass Spectrometry; D009578:Nitrobenzenes; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013629:Tamoxifen / Q000378:metabolism* / Q000494:pharmacology
D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D009578:Nitrobenzenes; D011506:Proteins; D013629:Tamoxifen; C002732:2-nitrobenzenesulfenyl chloride