Analysis of native or endogenous peptides in biofluids can provide valuable insight into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for noninvasive monitoring of human diseases. The noninvasive nature of urine collection and the abundance of peptides in the urine make analysis by high-throughput “peptidomics” methods an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regard to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements, in that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS-based peptidome analysis. We report on a novel adaptation of the standard solid-phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS-based peptidomics, in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. The mSPE method provides significantly improved efficiencies for the preparation of samples from urine. The mSPE method is found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis, due to optimized sample cleanup that provides improved experimental inference from confidently identified peptides.
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This chapter is based on research performed as part of an American Recovery and Reinvestment (ARRA)-funded project under Award Number U0163594 (to P Heeger), from the National Institute of Allergy and Infectious Diseases. The work was carried out by members of the Clinical Trials in Organ Transplantation (CTOT) and Clinical Trials in Organ Transplantation in Children (CTOT-C) consortia. The experimental work described herein was performed in the Environmental Molecular Sciences Laboratory (EMSL), a US Department of Energy (DOE) national scientific user facility located at PNNL in Richland, Washington, and in the Sarwal Lab at Stanford University and California Pacific Medical Center Research Institute. PNNL is a multi-program national laboratory operated by Battelle Memorial Institute for the DOE under Contract DE-AC05-76RL01830.
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