Faculty Collaboration Database

Identification of proteins in slow continuous ultrafiltrate by reversed-phase chromatography and proteomics. J Proteome Res 2004;3(6):1254-60

Date

12/15/2004

Pubmed ID

15595735

DOI

10.1021/pr0498640

Scopus ID

2-s2.0-11144302513 (requires institutional sign-in at Scopus site)   31 Citations

Abstract

Continuous modes of renal replacement therapy (CRRT) are increasingly being utilized in the intensive care unit. The removal of cytokines and other inflammatory proteins during ultrafiltration may be responsible for some of the beneficial effects of CRRT. We used proteomic tools to identify proteins found in the ultrafiltrate from a patient with acute renal failure. Identification of these proteins could help elucidate the mechanism(s) of improved outcome with continuous renal replacement therapy. Protein was loaded on a reversed-phase C4 column and eluted with stepwise isocratic flows starting with 0%, 5%, 10%, 25%, and 50% of acetonitrile. Effluent was collected, pooled, desalted, and separated by two-dimensional gel electrophoresis (2DE). Reversed-phase separation improved the resolution and the number of spots seen on the gels. Protein spots were digested with trypsin and spotted onto MALDI plates. Proteins were identified by either peptide mass fingerprinting using a MALDI-TOF mass spectrometer or by peptide sequencing using a MALDI-TOF/TOF tandem mass spectrometer. From 196 spots cut, 47 were identified, representing multiple charge forms of 10 different proteins. Proteins identified were albumin, apolipoprotein A-IV, beta-2-microglobulin, lithostathine, mannose-binding lectin associated serine protease 2 associated protein, plasma retinol-binding protein, transferrin, transthyretin, vitamin D-binding protein and Zn alpha-2 glycoprotein. Continuous renal replacement therapy is frequently used in acutely ill patients with renal failure. Removal of proteins occurs during this process. The physiological significance of this protein removal is unclear. Identification of these proteins will lead to better understanding of the role of protein removal in continuous renal replacement therapy.

Author List

Lefler DM, Pafford RG, Black NA, Raymond JR, Arthur JM

Author

John R. Raymond MD President, CEO, Professor in the President department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Chromatography, High Pressure Liquid
Dialysis Solutions
Electrophoresis, Gel, Two-Dimensional
Hemofiltration
Humans
Proteins
Proteomics
Renal Replacement Therapy
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization