title: A proteomic evaluation of urinary changes associated with cardiopulmonary bypass.
abstract: BACKGROUND
  The urinary proteome of patients undergoing cardiopulmonary bypass (CPB) may provide important insights into systemic and renal changes associated with the procedure. Such information may ultimately provide a basis to differentiate changes or properties associated with the development of acute kidney injury. While mass spectrometry (MS) analysis offers the potential for in-depth compositional analysis it is often limited in coverage and relative quantitation capacity. The aim of this study was to develop a process flow for the preparation and comparison of the intraoperative urinary proteome.
  
  
  METHODS
  Urines were collected from patients at the start of CPB and 1-h into CPB. Pooled samples (n = 5) from each time point were processed using a modified Filter Assisted Sample Preparation protocol. The resulting peptides were analyzed by 2D-LC-MS/MS and by 1D-LC-MS/MS SWATH (Sequential Window acquisition of All Theoretical fragment ion spectra).
  
  
  RESULTS
  The 2D-LC-MS/MS analysis identified 1324 proteins in the two pools, of which 744 were quantifiable. The SWATH approach provided quantitation for 730 proteins, 552 of which overlapped with the common population from the 2D-IDA results. Intensity correlation filtering between the two methods gave 475 proteins for biological interpretation. Proteins displaying greater than threefold changes (>log2 1.59) at 1-hour CPB relative to the initiation of CPB (26 down-regulated and 22 up-regulated) were selected for further analysis. Up-regulated proteins were enriched in GO terms related to humoral immune response, predominantly innate immunity (C4b, lactotransferrin, protein S100-A8, cathelicidin, myeloperoxidase) and extracellular matrix reorganization (e.g. MMP-9).
  
  
  CONCLUSIONS
  This study describes a scheme for processing urine from patients undergoing CPB for mass spectrometry-based analysis. The introduction of SWATH into the workflow offers a sample and instrument sparing approach to obtaining consistent in-depth sample analysis. The design of the methodology is such that it can be readily applied to large numbers of clinical samples with the potential for automation. The results also suggest that activation of the innate immune responses occur during cardiac bypass surgery.
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba and Health Sciences Centre, Room 799, John Buhler Research Center, 715 Mc Dermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Department of Internal Medicine, Section of Biomedical Proteomics, University of Manitoba, Winnipeg, MB Canada.
authors.forename: Ravi C
authors.initials: RC
authors.lastname: Dwivedi
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba and Health Sciences Centre, Room 799, John Buhler Research Center, 715 Mc Dermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Department of Internal Medicine, Section of Biomedical Proteomics, University of Manitoba, Winnipeg, MB Canada.
authors.forename: Mario
authors.initials: M
authors.lastname: Navarrete
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba and Health Sciences Centre, Room 799, John Buhler Research Center, 715 Mc Dermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Department of Internal Medicine, Section of Biomedical Proteomics, University of Manitoba, Winnipeg, MB Canada ; Cardiac Sciences Program, St. Boniface Hospital Research Centre, Winnipeg, Canada.
authors.forename: Nora
authors.initials: N
authors.lastname: Choi
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba and Health Sciences Centre, Room 799, John Buhler Research Center, 715 Mc Dermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Department of Internal Medicine, Section of Biomedical Proteomics, University of Manitoba, Winnipeg, MB Canada.
authors.forename: Victor
authors.initials: V
authors.lastname: Spicer
authors.affiliation: Department of Internal Medicine, Section of Nephrology, University of Manitoba, Winnipeg, MB Canada.
authors.forename: Claudio
authors.initials: C
authors.lastname: Rigatto
authors.affiliation: Department of Surgery, University of Manitoba, Winnipeg, MB Canada ; Cardiac Sciences Program, St. Boniface Hospital Research Centre, Winnipeg, Canada.
authors.forename: Rakesh C
authors.initials: RC
authors.lastname: Arora
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba and Health Sciences Centre, Room 799, John Buhler Research Center, 715 Mc Dermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Department of Internal Medicine, Section of Biomedical Proteomics, University of Manitoba, Winnipeg, MB Canada.
authors.forename: Oleg
authors.initials: O
authors.lastname: Krokhin
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba and Health Sciences Centre, Room 799, John Buhler Research Center, 715 Mc Dermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Department of Internal Medicine, Section of Biomedical Proteomics, University of Manitoba, Winnipeg, MB Canada ; Department of Internal Medicine, Section of Nephrology, University of Manitoba, Winnipeg, MB Canada ; Department of Immunology, University of Manitoba, Winnipeg, MB Canada.
authors.forename: Julie
authors.initials: J
authors.lastname: Ho
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba and Health Sciences Centre, Room 799, John Buhler Research Center, 715 Mc Dermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Department of Internal Medicine, Section of Biomedical Proteomics, University of Manitoba, Winnipeg, MB Canada.
authors.forename: John A
authors.initials: JA
authors.lastname: Wilkins
keywords: Cardiac surgery; Data dependent acquisition (DDA); Data independent acquisition (DIA); Information dependent data acquisition (IDA); Label free quantitation; Molecular weight cut off (MWCO) filters; Proteomics; Renal; SWATH; Urine