title: Multiplexed quantification of estrogen receptor and HER2/Neu in tissue and cell lysates by peptide immunoaffinity enrichment mass spectrometry.
abstract: Access to a wider range of quantitative protein assays would significantly impact the number and use of tissue markers in guiding disease treatment. Quantitative mass spectrometry-based peptide and protein assays, such as immuno-SRM assays, have seen tremendous growth in recent years in application to protein quantification in biological fluids such as plasma or urine. Here, we extend the capability of the technique by demonstrating the application of a multiplexed immuno-SRM assay for quantification of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) levels in cell line lysates and human surgical specimens. The performance of the assay was characterized using peptide response curves, with linear ranges covering approximately four orders of magnitude and limits of detection in the low fmol/mg lysate range. Reproducibility was acceptable with median coefficients of variation of approximately 10%. We applied the assay to measurements of ER and HER2 in well-characterized cell line lysates with good discernment based on ER/HER2 status. Finally, the proteins were measured in surgically resected breast cancers, and the results showed good correlation with ER/HER2 status determined by clinical assays. This is the first implementation of the peptide-based immuno-SRM assay technology in cell lysates and human surgical specimens.
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
authors.forename: Regine M
authors.initials: RM
authors.lastname: Schoenherr
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Whiteaker
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.forename: Richard G
authors.initials: RG
authors.lastname: Ivey
authors.forename: Mary
authors.initials: M
authors.lastname: Trute
authors.forename: Jacob
authors.initials: J
authors.lastname: Kennedy
authors.forename: Uliana J
authors.initials: UJ
authors.lastname: Voytovich
authors.forename: Ping
authors.initials: P
authors.lastname: Yan
authors.forename: Chenwei
authors.initials: C
authors.lastname: Lin
authors.forename: Amanda G
authors.initials: AG
authors.lastname: Paulovich
mesh_terms: D000595:Amino Acid Sequence; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000378:metabolism; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000378:metabolism; D002138:Calibration; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D005260:Female; D057166:High-Throughput Screening Assays; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis* / Q000378:metabolism; D011960:Receptors, Estrogen / Q000032:analysis* / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D014408:Biomarkers, Tumor; D002457:Cell Extracts; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; C508053:ERBB2 protein, human