P56
A NOVEL APPROACH TO URINE MASS SPECTROMETRY WITHOUT ABUNDANT
PROTEIN DEPLETION USING A UROMODULIN EXCLUSION LIST
Hiemstra, T¹, Hester, S², Karet, F¹, Lilley, K²
¹Department of Medical Genetics and ²Cambridge Centre for Proteomics, University of Cambridge
INTRODUCTION: Recent advances in proteomics have been followed by many urinary biomarker
studies utilising mass spectrometry. However, the presence of abundant proteins in mass spectrometry
samples leads to repeated sampling of abundant peptides, potential ion suppression and reduction in
sampling of less abundant peptide spectra. For urinary studies, most investigators advocate removal of the
abundant urinary protein uromodulin to improve resolution for low abundance protein discovery. This
approach may lead to concomitant removal of proteins of interest. We describe a novel approach to
improve resolution of proteins identified from human urinary exosomal samples by LC-MS/MS that
negates abundant protein removal.
METHODS: Exosomes were isolated from the urine of a healthy male volunteer by differential
centrifugation. Following ammonium acetate precipitation, samples were fractionated by 1D SDS-PAGE,
in-gel trypsin digested, and subjected to LC-MS/MS on a high mass accuracy LTQ-Orbitrap instrument.
Spectra were analysed using MASCOT-PERCOLATOR (a novel algorithm incorporating machine
learning), and searched against the human IPI protein database (v4.3) setting a False Discovery Rate
(FDR) of 0.1. All spectra representing uromodulin peptides were identified and ranked by MASCOT
peptide score. The 20 most intense spectra were identified and corresponding mass-to-charge ratios (m/z)
noted. Samples were re-run and abundant uromodulin peptide spectra were rejected by forced exclusion
of listed m/z, specifying an exclusion window by ‘percent of reference mass’ using Xcalibur software.
RESULTS: Overall, 369 proteins were identified using the conventional approach. Application of the
uromodulin exclusion list reduced the MASCOT protein score for uromodulin from 1211 to 468, and the
number of peptides attributed to uromodulin from 23 to 7. However, total protein identification fell to 99
by this method. Importantly, 43 of these had not been identified without the exclusion list. By combining
these analyses, the total number of identifications was therefore increased to 412, representing a 12%
increase in proteins.
CONCLUSION: Application of an exclusion list for uromodulin allows identification of proteins not
identified by conventional methods, but excludes many proteins only identified by conventional methods.
Our results indicate that application of an exclusion list for uromodulin cannot be used in isolation but,
when used in parallel with conventional analysis, provides an attractive alternative to abundant protein
removal.