title: Combining filter-aided sample preparation and pseudoshotgun technology to profile the proteome of a low number of early passage human melanoma cells.
abstract: The performance of two proteomic sample preparation methods, "pseudoshotgun" (PSG) and filter-aided sample preparation (FASP) were compared in terms of the number of identified proteins, representation of cellular component GO (gene ontology) categories in the obtained list of proteins, and the efficiency of both methods in the proteomic analysis of a very low number of cells. Both methods were combined to obtain a proteomic profile of a short-term culture (passage 3) of melanoma cells, established in our laboratory from a human metastatic melanoma lesion. The data revealed that with FASP, usually more proteins are identified than with PSG when analyzing a higher number of cells (≥ 5000/injection), whereas PSG is favorable when analyzing only a very small amount of cells (250-500/injection). PSG and FASP, however, are complementary techniques, as combining both methods further increases the number of identified proteins. Moreover, we show that it is feasible to identify a substantial number of proteins from only 250 cells/injection that is equivalent to 60 ng of protein.
authors.affiliation: Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.
authors.forename: Margarita
authors.initials: M
authors.lastname: Maurer
authors.forename: André C
authors.initials: AC
authors.lastname: Müller
authors.forename: Christine
authors.initials: C
authors.lastname: Wagner
authors.forename: Marie L
authors.initials: ML
authors.lastname: Huber
authors.forename: Elena L
authors.initials: EL
authors.lastname: Rudashevskaya
authors.forename: Stephan N
authors.initials: SN
authors.lastname: Wagner
authors.forename: Keiryn L
authors.initials: KL
authors.lastname: Bennett
mesh_terms: D002452:Cell Count; D002853:Chromatography, Liquid; D005374:Filtration / Q000379:methods; D006801:Humans; D057230:Limit of Detection; D008545:Melanoma / Q000737:chemistry* / Q000556:secondary; D009363:Neoplasm Proteins / Q000737:chemistry / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000302:isolation & purification*; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification*; D012878:Skin Neoplasms / Q000737:chemistry* / Q000473:pathology; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry; D014407:Tumor Cells, Cultured
chemical_list: D009363:Neoplasm Proteins; D010446:Peptide Fragments; D020543:Proteome; D014357:Trypsin