title: Protocol: MYTHBUSTERS: a universal procedure for sample preparation for mass spectrometry.
abstract: Improvements in proteomic strategies from the development of new and robust separation and identification techniques have led to broad applications of proteomics to solve numerous biological questions. For all analyses, sample quality is unquestionably a critical factor; therefore protein extraction is of outmost importance. The ideal extraction method should provide reproducible spectra of the most comprehensive repertoire of proteins, while minimizing sample loss and degradation. It is already known that to capture the whole proteome is an unenforceable task. Many protein extraction protocols have been described, yet there is no "one perfect procedure" taking into account the vast diversity of biological and physical properties of proteins, including their charge, size, hydrophobicity, interactions and sub-cellular localization. The research presented here reflects the main obstacle occurring in proteomic experimental design; i.e. the lack of reproducibility as a result of alterations in protein extraction methods. We have performed a series of experiments, aimed towards identification of the aptamer-binding partners in cancerous cells. Aptamers are chemically synthesized, short, single-stranded nucleic acids with a strictly defined three-dimensional structure, which allows them to interact with a target molecule with high affinity. The low immunogenicity and cellular- targeting properties of aptamers might facilitate design of suitable drugs with low side-effects. Aptamers can be used for identification of molecules associated with a pathogenic state of a cell. Aptamers can be considered as a powerful tool, since they possess unique properties to benefit cancer diagnosis, prevention and treatment. We have used different types of protein extraction methods prior to analyses of complex biological samples by mass spectrometry, based on slight changes of homogenization buffers, and have observed the changes in the identified compounds. These results should prove to be very useful for future proteomic studies and the design of studies in terms of sample preparation, especially sample homogenization and protein extraction.
authors.affiliation: Department of Biochemistry and Neurobiology, AGH University of Science and Technology, Mickiewicza 30, 30-059 Krakow, Poland. drabik@agh.edu.pl.
authors.forename: Anna
authors.initials: A
authors.lastname: Drabik
authors.affiliation: Department of Biochemistry and Neurobiology, AGH University of Science and Technology, Mickiewicza 30, 30-059 Krakow, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Ner-Kluza
authors.affiliation: Department of Biochemistry and Neurobiology, AGH University of Science and Technology, Mickiewicza 30, 30-059 Krakow, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Bodzon-Kulakowska
authors.affiliation: Department of Biochemistry and Neurobiology, AGH University of Science and Technology, Mickiewicza 30, 30-059 Krakow, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Suder
mesh_terms: D052158:Aptamers, Peptide / Q000032:analysis / Q000737:chemistry*; D045744:Cell Line, Tumor; D017408:Guidelines as Topic; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000592:standards*; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry*; D011471:Prostatic Neoplasms / Q000737:chemistry*; D025941:Protein Interaction Mapping / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000592:standards*
chemical_list: D052158:Aptamers, Peptide; D009363:Neoplasm Proteins