title: A two-stage spin cartridge for integrated protein precipitation, digestion and SDS removal in a comparative bottom-up proteomics workflow.
abstract: Protein precipitation with organic solvent is an effective means of depleting contaminants such as sodium dodecyl sulfate (SDS), while maintaining high analyte recovery. Here, we report the use of a disposable two-stage spin cartridge to facilitate isolation of the precipitated protein, with subsequent enzyme digestion and peptide cleanup in the cartridge. An upper filtration cartridge retains over 95% of the protein (10 μg BSA), with 99.75% detergent depleted from a sample initially containing 2% SDS. Following precipitation, a plug attached to the base of the filtration cartridge retains the solution to enable tryptic digestion in the vial, while a solid phase extraction cartridge attached to the base of the filter facilitates peptide cleanup post-digestion. A GELFrEE fractionated Escherichia coli proteome extract processed with the spin cartridge yields similar protein identifications compared to controls (226 vs 216 for control), and with an increased number of unique peptides (1753 vs 1554 for control). The device is applied to proteome characterization of rat kidneys experiencing a surgically induced ureteral tract obstruction, revealing several statistically altered proteins, consistent with the morphology and expected pathophysiology of the disease.
  
  
  CONCLUSIONS
  Conventionally, protein precipitation involves extended centrifugation to pellet the sample, with careful pipetting to remove the supernatant without disturbing the pellet. The method is not only time consuming but is highly subject to the skill of the individual, particularly at lower protein concentrations where the pellet may not be visible. As such, protein precipitation is often overlooked in proteomics, favoring column-based approaches to concentrate or purify samples. Here, all aspects of sample manipulation are integrated into a simple disposable cartridge. The device enables SDS depletion, sample preconcentration, resolubilization, derivatization, digestion, and peptide cleanup in a highly repeatable and easily multiplexed format. The device is ideally suited for comparative proteome studies. Antenatal hydronephrosis is a congenital disorder affecting 1-5% of all pregnancies, and can require surgical intervention to avoid loss of renal function. Using our device, we investigated the impact of hydronephrosis on the kidneys in a surgically induced animal model of the disease. Proteome analysis points to decreased metabolic activity in the obstructed kidney, with upregulation of proteins involved in cytoskeletal organization. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.
authors.affiliation: Department of Chemistry, Dalhousie University, Canada.
authors.forename: Andrew M J
authors.initials: AM
authors.lastname: Crowell
authors.affiliation: Department of Urology and Pathology, Dalhousie University, Canada.
authors.forename: Dawn L
authors.initials: DL
authors.lastname: MacLellan
authors.affiliation: Department of Chemistry, Dalhousie University, Canada. Electronic address: Alan.doucette@dal.ca.
authors.forename: Alan A
authors.initials: AA
authors.lastname: Doucette
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006869:Hydronephrosis / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; D020543:Proteome; D012967:Sodium Dodecyl Sulfate
keywords: Antenatal hydronephrosis; Automation; Kidney; Protein precipitation; SDS depletion; Spin cartridge