title: Rapid Protein Digestion and Purification with Membranes Attached to Pipet Tips.
abstract: This paper presents rapid protein purification and proteolysis methods that integrate membrane technology and pipet tips. Pushing a protein-containing solution through a protease-modified membrane at the end of a pipet tip digests proteins in 30 s or less, and the short proteolysis time avoids reformation of disulfide bonds to enable tryptic digestion without alkylation of cysteine residues. Moreover, proteolysis is more complete than digestion for 30 min in solution. Antibody digestion at the end of a pipet tip leads to 100% peptide coverage in MS analyses. Similarly, when membranes contain Ni(2+) complexes, pipetting aqueous polyhistidine-tagged protein through the membrane and subsequent rinsing and elution yield purified polyhistidine-tagged protein in 2 min. These applications demonstrate the potential for combining functional membranes and pipet tips for rapid sample purification and pretreatment.
authors.affiliation: Department of Chemistry, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Ning
authors.affiliation: Department of Chemistry, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Merlin L
authors.initials: ML
authors.lastname: Bruening
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002623:Chemistry Techniques, Analytical / Q000379:methods* / Q000592:standards; D056831:Coordination Complexes / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D009532:Nickel / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D059748:Proteolysis; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
chemical_list: D056831:Coordination Complexes; D008567:Membranes, Artificial; D011506:Proteins; D009532:Nickel; D010447:Peptide Hydrolases; D014357:Trypsin