title: MStern Blotting-High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates.
abstract: We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. In summary, our new method maintains the strengths of FASP and simultaneously overcomes one of the major limitations of FASP without compromising protein identification and quantification.
authors.affiliation: From the ‡Proteomics Center and Department of Pathology and ¶Department of Pathology and F.M. Kirby Neurobiology Center, Boston Children's Hospital and Department of Neurology, Harvard Medical School, Boston, Massachusetts.
authors.forename: Sebastian T
authors.initials: ST
authors.lastname: Berger
authors.affiliation: From the ‡Proteomics Center and Department of Pathology and ¶Department of Pathology and F.M. Kirby Neurobiology Center, Boston Children's Hospital and Department of Neurology, Harvard Medical School, Boston, Massachusetts.
authors.forename: Saima
authors.initials: S
authors.lastname: Ahmed
authors.affiliation: From the ‡Proteomics Center and Department of Pathology and ¶Department of Pathology and F.M. Kirby Neurobiology Center, Boston Children's Hospital and Department of Neurology, Harvard Medical School, Boston, Massachusetts.
authors.forename: Jan
authors.initials: J
authors.lastname: Muntel
authors.affiliation: From the ‡Proteomics Center and Department of Pathology and ¶Department of Pathology and F.M. Kirby Neurobiology Center, Boston Children's Hospital and Department of Neurology, Harvard Medical School, Boston, Massachusetts, **Department of Veterinary Science and Public Health, Università degli Studi di Milano, Italy;
authors.forename: Nerea
authors.initials: N
authors.lastname: Cuevas Polo
authors.affiliation: §Division of Emergency Medicine, Boston Children's Hospital, Boston, Massachusetts.
authors.forename: Richard
authors.initials: R
authors.lastname: Bachur
authors.affiliation: ‡‡Molecular Pharmacology & Chemistry Program, Sloan Kettering Institute, Department of Pediatrics, Memorial Sloan Kettering Cancer Center and Weill Medical College of Cornell University, New York, New York.
authors.forename: Alex
authors.initials: A
authors.lastname: Kentsis
authors.forename: Judith
authors.initials: J
authors.lastname: Steen
authors.affiliation: From the ‡Proteomics Center and Department of Pathology and ¶Department of Pathology and F.M. Kirby Neurobiology Center, Boston Children's Hospital and Department of Neurology, Harvard Medical School, Boston, Massachusetts, Hanno.Steen@childrens.harvard.edu.
authors.forename: Hanno
authors.initials: H
authors.lastname: Steen
mesh_terms: D000327:Adsorption; D015415:Biomarkers / Q000652:urine; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008567:Membranes, Artificial; D010048:Ovarian Cysts / Q000652:urine; D011145:Polyvinyls / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D013048:Specimen Handling; D005465:Fluorocarbon Polymers
chemical_list: D015415:Biomarkers; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers