Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology.
An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17beta-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of <4%. The reproducibility of protein identifications was approximately 60%-67% between duplicate, and approximately 50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P < 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate approximately 2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, approximately 16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up- or down-regulated.
Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Jenny M
JM
Armenta
Ina
I
Hoeschele
Iulia M
IM
Lazar
D014408:Biomarkers, Tumor / Q000032:analysis / Q000096:biosynthesis; D001943:Breast Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D009363:Neoplasm Proteins / Q000032:analysis / Q000096:biosynthesis; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D013629:Tamoxifen / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods*
D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D011506:Proteins; D013629:Tamoxifen; D004958:Estradiol