Proteomics pipeline for biomarker discovery of laser capture microdissected breast cancer tissue.
Mass spectrometry (MS)-based label-free proteomics offers an unbiased approach to screen biomarkers related to disease progression and therapy-resistance of breast cancer on the global scale. However, multi-step sample preparation can introduce large variation in generated data, while inappropriate statistical methods will lead to false positive hits. All these issues have hampered the identification of reliable protein markers. A workflow, which integrates reproducible and robust sample preparation and data handling methods, is highly desirable in clinical proteomics investigations. Here we describe a label-free tissue proteomics pipeline, which encompasses laser capture microdissection (LCM) followed by nanoscale liquid chromatography and high resolution MS. This pipeline routinely identifies on average ∼10,000 peptides corresponding to ∼1,800 proteins from sub-microgram amounts of protein extracted from ∼4,000 LCM breast cancer epithelial cells. Highly reproducible abundance data were generated from different technical and biological replicates. As a proof-of-principle, comparative proteome analysis was performed on estrogen receptor α positive or negative (ER+/-) samples, and commonly known differentially expressed proteins related to ER expression in breast cancer were identified. Therefore, we show that our tissue proteomics pipeline is robust and applicable for the identification of breast cancer specific protein markers.
Department of Medical Oncology and Daniel Den Hoed Cancer Center, Erasmus University Medical Center, Dr. Molewaterplein 50, Rotterdam, the Netherlands. n.liu@erasmusmc.nl
Ning Qing
NQ
Liu
René B H
RB
Braakman
Christoph
C
Stingl
Theo M
TM
Luider
John W M
JW
Martens
John A
JA
Foekens
Arzu
A
Umar
D014408:Biomarkers, Tumor / Q000737:chemistry / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002469:Cell Separation / Q000379:methods; D015536:Down-Regulation; D004848:Epithelium / Q000378:metabolism / Q000473:pathology; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D061346:Laser Capture Microdissection; D042361:Mammary Glands, Human / Q000378:metabolism* / Q000473:pathology; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011960:Receptors, Estrogen / Q000737:chemistry / Q000378:metabolism; D011980:Receptors, Progesterone / Q000737:chemistry / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D010455:Peptides; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone