利用iTRAQ化學標定方法進行乳癌細胞系MCF-7多重抗藥性之差異蛋白質體學分析
Abstract
乳癌是全世界女性中最常見的癌症類型，在台灣，乳癌為女性癌症的好發率首位，死亡率則位居第四。乳癌荷爾蒙治療是一種具有全身療效而且副作用相對最少的療法，許多荷爾蒙藥物已成功的用於轉移性乳癌或手術後輔助治療上，並且都有優異的療效。本篇研究使用同重元素相對及絕對定量(iTRAQ)的化學標記方法搭配質譜技術，分析乳癌細胞系MCF-7經由乳癌荷爾蒙藥物(Tamoxifen)，以及常搭配的標靶藥物(Everolimus、Palbociclib) 治療後，其蛋白質產生的相對變化量。為了降低樣品的複雜度，iTRAQ標定的胜肽樣品會先經由液相等電聚焦法(sIEF)、鹼性逆相層析法(BRP)、強陽離子交換層析法(SCX)做第一維的分離，再進行奈米級液相層析連接質譜儀進行分析。於三次生物性重複實驗之中，共鑑定到 8855 個蛋白質以及61169個不重複的胜肽，並比較了三種分離方法鑑定結果的正交性與互補性。在Tamoxifen、Everolimus及Palbociclib相對定量出的蛋白質中，分別挑選出了362、339、827個差異蛋白質，並以Metascape網站進行pathway分類比較，主要與beta-oxidation of pristanoyl-CoA, nucleoside phosphate metabolic process, metabolism of lipids有較大的相關性，並從中挑選出幾個蛋白質(Q96B36-2、O95817、P11279-2、O76070、E9PC90、P11279-2)做進一步探討，期望能發現新穎的蛋白質，日後可能做為乳癌荷爾蒙治療及標靶治療的生物標記分子，對乳癌的發病機理和後續臨床疾病之診斷治療提供新的方向。
Breast cancer is the most common cancer among women worldwide. It is also the most commonly occurring cancer and ranks as the fourth leading cause of death in Taiwan. Hormonal therapy is a form of systemic therapy with relatively few side effects. Hormone therapies have been successfully used in metastatic breast cancer or postoperative adjuvant therapy for patients and has excellent efficacy. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) was used to investigate the differential protein profiles in MCF-7 cell lines treated with hormone therapy drug (Tamoxifen) and targeted therapy drugs (Everolimus, Palbociclib). In order to reduce the complexity of the samples, the iTRAQ labeled peptides were fractionated by solution isoelectric focusing (sIEF), strong cation exchange chromatography (SCX) or basic reverse phase chromatography (BRP), followed by nano-flow liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis. A total of 8855 proteins and 61169 unique peptides were successfully identified in three biological replicates, and the orthogonality and complementarity of three fractionations were assessed. Moreover, 362, 339 and 827 differential proteins were selected in tamoxifen, everolimus and palbociclib -resistant cells , respectively. These differential proteins were found to be mainly associated with beta-oxidation of pristanoyl-CoA, nucleoside phosphate metabolic process, and metabolism of lipids. Some of these differential proteins (Q96B36-2、O95817、P11279-2、O76070、E9PC90、P11279-2) were selected as candidates and could serve as potential novel diagnostic biomarkers for breast cancer therapy.
Breast cancer is the most common cancer among women worldwide. It is also the most commonly occurring cancer and ranks as the fourth leading cause of death in Taiwan. Hormonal therapy is a form of systemic therapy with relatively few side effects. Hormone therapies have been successfully used in metastatic breast cancer or postoperative adjuvant therapy for patients and has excellent efficacy. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) was used to investigate the differential protein profiles in MCF-7 cell lines treated with hormone therapy drug (Tamoxifen) and targeted therapy drugs (Everolimus, Palbociclib). In order to reduce the complexity of the samples, the iTRAQ labeled peptides were fractionated by solution isoelectric focusing (sIEF), strong cation exchange chromatography (SCX) or basic reverse phase chromatography (BRP), followed by nano-flow liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis. A total of 8855 proteins and 61169 unique peptides were successfully identified in three biological replicates, and the orthogonality and complementarity of three fractionations were assessed. Moreover, 362, 339 and 827 differential proteins were selected in tamoxifen, everolimus and palbociclib -resistant cells , respectively. These differential proteins were found to be mainly associated with beta-oxidation of pristanoyl-CoA, nucleoside phosphate metabolic process, and metabolism of lipids. Some of these differential proteins (Q96B36-2、O95817、P11279-2、O76070、E9PC90、P11279-2) were selected as candidates and could serve as potential novel diagnostic biomarkers for breast cancer therapy.
Description
Keywords
差異蛋白質, 乳癌, iTRAQ, differential protein, breast cancer