title: Intact protein profiling in breast cancer biomarker discovery: protein identification issue and the solutions based on 3D protein separation, bottom-up and top-down mass spectrometry.
abstract: Proteomics profiling of intact proteins based on MALDI-TOF MS and derived platforms has been used in cancer biomarker discovery studies. This approach suffers from a number of limitations such as low resolution, low sensitivity, and that no knowledge is available on the identity of the respective proteins in the discovery mode. Nevertheless, it remains the most high-throughput, untargeted mode of clinical proteomics studies to date. Here we compare key protein separation and MS techniques available for protein biomarker identification in this type of studies and define reasons of uncertainty in protein peak identity. As a result of critical data analysis, we consider 3D protein separation and identification workflows as optimal procedures. Subsequently, we present a new protocol based on 3D LC-MS/MS with top-down at high resolution that enabled the identification of HNRNP A2/B1 intact peptide as correlating with the estrogen receptor expression in breast cancer tissues. Additional development of this general concept toward next generation, top-down based protein profiling at high resolution is discussed.
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic. bouchal@chemi.muni.cz
authors.forename: Pavel
authors.initials: P
authors.lastname: Bouchal
authors.forename: Monika
authors.initials: M
authors.lastname: Dvorakova
authors.forename: Alexander
authors.initials: A
authors.lastname: Scherl
authors.forename: Spiros D
authors.initials: SD
authors.lastname: Garbis
authors.forename: Rudolf
authors.initials: R
authors.lastname: Nenutil
authors.forename: Borivoj
authors.initials: B
authors.lastname: Vojtesek
mesh_terms: D000595:Amino Acid Sequence; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism*; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D010455:Peptides