title: Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis.
abstract: Despite recent developments in bottom-up proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS-based protein extraction with rapid detergent removal, reactor-type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack-this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow-through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential proteomics tool.
authors.affiliation: Cancer Research UK Centre, Leeds Institute of Cancer and Pathology, St James's University Hospital, Leeds, UK.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Zougman
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Selby
authors.forename: Rosamonde E
authors.initials: RE
authors.lastname: Banks
mesh_terms: D003902:Detergents / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D047468:Immunoprecipitation; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
chemical_list: D003902:Detergents; D010446:Peptide Fragments; D011506:Proteins; D012967:Sodium Dodecyl Sulfate
keywords: Proteomics sample preparation; Technology