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.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
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.
Shimwell NJ et al (2013) Combined proteome and transcriptome analyses for the discovery of urinary biomarkers for urothelial carcinoma. Br J Cancer 108(9):1854–1861CrossRefPubMedPubMedCentralGoogle Scholar
Albalat A, Mischak H, Mullen W (2011) Clinical application of urinary proteomics/peptidomics. Expert Rev Proteomics 8(5):615–629CrossRefPubMedGoogle Scholar
Machtejevas E et al (2009) Profiling of endogenous peptides by multidimensional liquid chromatography: on-line automated sample cleanup for biomarker discovery in human urine. J Sep Sci 32(13):2223–2232CrossRefPubMedGoogle Scholar
Schulz-Knappe P et al (2001) Peptidomics: the comprehensive analysis of peptides in complex biological mixtures. Comb Chem High Throughput Screen 4(2):207–217CrossRefPubMedGoogle Scholar
Cutillas PR et al (2003) Detection and analysis of urinary peptides by on-line liquid chromatography and mass spectrometry: application to patients with renal Fanconi syndrome. Clin Sci (Lond) 104(5):483–490CrossRefGoogle Scholar
Wittke S et al (2005) Discovery of biomarkers in human urine and cerebrospinal fluid by capillary electrophoresis coupled to mass spectrometry: towards new diagnostic and therapeutic approaches. Electrophoresis 26(7–8):1476–1487CrossRefPubMedGoogle Scholar
Schaub S et al (2004) Urine protein profiling with surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry. Kidney Int 65(1):323–332CrossRefPubMedGoogle Scholar
Sigdel TK, Klassen RB, Sarwal MM (2009) Interpreting the proteome and peptidome in transplantation. Adv Clin Chem 47:139–169CrossRefPubMedGoogle Scholar
Qian WJ et al (2005) Probability-based evaluation of peptide and protein identifications from tandem mass spectrometry and SEQUEST analysis: the human proteome. J Proteome Res 4(1):53–62CrossRefPubMedGoogle Scholar