title: Multiple reaction monitoring of multiple low-abundance transcription factors in whole lung cancer cell lysates.
abstract: Lung cancer-related transcription factors (TFs) were identified by integrating previously reported genomic, transcriptomic, and proteomic data and were quantified by multiple reaction monitoring (MRM) in various cell lines. All experiments were performed without affinity depletion or subfractionation of cell lysates. Since the target proteins were expected to be present in low abundance, we experimentally optimized MRM transition parameters with chemically synthesized peptides. Quantitation was based on stable isotope-labeled standard peptides (SIS peptides). Out of 288 MRM measurements (36 peptides representing 28 TFs × 8 cell lines), 241 were successfully obtained within a quantitation limit of 15 amol, 221 measurements (91.7%) showed coefficients of variation (CVs) of ≤ 20%, and 149 (61.8%) showed CVs of ≤ 10%, quantifying as low as 19.4 amol/μg protein for STAT2 with a CV of 6.3% in an A549 cell. Comparisons between MRM measurements and levels of the corresponding mRNAs revealed linear, nonlinear, or no relationship between protein and mRNA levels, indicating the need for an MRM assay. An integrative analysis of MRM and gene expression profiles from doxorubicin-resistant H69AR and sensitive H69 cells further showed that 14 differentially expressed TFs, such as STAT1 and SMAD4, regulated genes associated with drug resistance and cell differentiation-related processes. Thus, the analytical performance of MRM for the quantitation of low abundance TFs suggests its usefulness for biological application.
authors.affiliation: Theragnosis Research Center, Korea Institute of Science and Technology, Seoul, Korea.
authors.forename: Jun Seok
authors.initials: JS
authors.lastname: Kim
authors.forename: Youngju
authors.initials: Y
authors.lastname: Lee
authors.forename: Min Young
authors.initials: MY
authors.lastname: Lee
authors.forename: Jihye
authors.initials: J
authors.lastname: Shin
authors.forename: Jung Min
authors.initials: JM
authors.lastname: Han
authors.forename: Eun Gyeong
authors.initials: EG
authors.lastname: Yang
authors.forename: Myeong-Hee
authors.initials: MH
authors.lastname: Yu
authors.forename: Sunghoon
authors.initials: S
authors.lastname: Kim
authors.forename: Daehee
authors.initials: D
authors.lastname: Hwang
authors.forename: Cheolju
authors.initials: C
authors.lastname: Lee
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D002277:Carcinoma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D007553:Isotope Labeling; D057230:Limit of Detection; D008175:Lung Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000302:isolation & purification; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D051901:Smad4 Protein / Q000235:genetics* / Q000378:metabolism
chemical_list: D000903:Antibiotics, Antineoplastic; D002457:Cell Extracts; D009363:Neoplasm Proteins; D010455:Peptides; D012333:RNA, Messenger; C497796:SMAD4 protein, human; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D051901:Smad4 Protein; D004317:Doxorubicin