Weixian Zhou1, Feifei Xu1, Danni Li2, Yun Chen3,4. 1. School of Pharmacy, Nanjing Medical University, Nanjing, China. 2. Department of Lab Medicine and Pathology, University of Minnesota, Minneapolis, MN; ychen@njmu.edu.cn dannili@umn.edu. 3. School of Pharmacy, Nanjing Medical University, Nanjing, China; ychen@njmu.edu.cn dannili@umn.edu. 4. State Key Laboratory of Reproductive Medicine, Nanjing, China.
Abstract
BACKGROUND: Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is a particularly aggressive type of the disease. To date, much evidence has indicated that accurate HER2 status detection is crucial for prognosis and treatment strategy selection. Thus, bioanalytical techniques for early and accurate detection of HER2 have the potential to improve patient care. Currently, the widely used immunohistochemical staining normally has problems with reproducibility and lack of standardization, resulting in poor concordance between laboratories. Aptamers are a good alternative, but the extent of their use in quantitative analysis of HER2 is limited because of the lack of effective detection methods. METHODS: We developed a quasi-targeted proteomics assay and converted the HER2 signal into the mass response of reporter peptide by a combination of aptamer-peptide probe and LC-MS/MS. RESULTS: The selected aptamer-peptide probe consisted of aptamer HB5 and the substrate peptide GDKAVLGVDPFR that contained the reporter peptide AVLGVDPFR. After characterization of this newly synthesized probe (e.g., conjugation efficiency, stability, binding affinity, specificity, and digestion efficiency), probe binding and trypsin shaving conditions were optimized. The resulting limit of quantification for HER2 was 25 pmol/L. Then, the quasi-targeted proteomics assay was applied to determine the HER2 concentrations in the HER2-positive breast cancer cells BT474 and SK-BR-3, the HER2-negative breast cancer cells MDA-MB-231 and MCF-7, and 36 pairs of human breast primary tumors and adjacent normal tissue samples. The results were highly concordant with those obtained by immunohistochemistry with reflex testing by fluorescent in situ hybridization. CONCLUSIONS: Quasi-targeted proteomics can be a quantitative alternative for HER2 detection.
BACKGROUND:Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is a particularly aggressive type of the disease. To date, much evidence has indicated that accurate HER2 status detection is crucial for prognosis and treatment strategy selection. Thus, bioanalytical techniques for early and accurate detection of HER2 have the potential to improve patient care. Currently, the widely used immunohistochemical staining normally has problems with reproducibility and lack of standardization, resulting in poor concordance between laboratories. Aptamers are a good alternative, but the extent of their use in quantitative analysis of HER2 is limited because of the lack of effective detection methods. METHODS: We developed a quasi-targeted proteomics assay and converted the HER2 signal into the mass response of reporter peptide by a combination of aptamer-peptide probe and LC-MS/MS. RESULTS: The selected aptamer-peptide probe consisted of aptamer HB5 and the substrate peptide GDKAVLGVDPFR that contained the reporter peptide AVLGVDPFR. After characterization of this newly synthesized probe (e.g., conjugation efficiency, stability, binding affinity, specificity, and digestion efficiency), probe binding and trypsin shaving conditions were optimized. The resulting limit of quantification for HER2 was 25 pmol/L. Then, the quasi-targeted proteomics assay was applied to determine the HER2 concentrations in the HER2-positive breast cancer cells BT474 and SK-BR-3, the HER2-negative breast cancer cells MDA-MB-231 and MCF-7, and 36 pairs of humanbreast primary tumors and adjacent normal tissue samples. The results were highly concordant with those obtained by immunohistochemistry with reflex testing by fluorescent in situ hybridization. CONCLUSIONS: Quasi-targeted proteomics can be a quantitative alternative for HER2 detection.
Authors: Sergey Radko; Konstantin Ptitsyn; Svetlana Novikova; Yana Kiseleva; Alexander Moysa; Leonid Kurbatov; Maria Mannanova; Victor Zgoda; Elena Ponomarenko; Andrey Lisitsa; Alexander Archakov Journal: Biomedicines Date: 2020-05-24