Current state of the art for enhancing urine biomarker discovery

Michael Harpole, Justin Davis, Virginia Espina
2016 Espert Review of Proteomics  
Urine is a highly desirable biospecimen for biomarker analysis because it can be collected recurrently by non-invasive techniques, in relatively large volumes. Urine contains cellular elements, biochemicals, and proteins derived from glomerular filtration of plasma, renal tubule excretion, and urogenital tract secretions that reflect, at a given time point, an individual's metabolic and pathophysiologic state. High-resolution mass spectrometry, coupled with state of the art fractionation
more » ... are revealing the plethora of diagnostic/prognostic proteomic information existing within urinary exosomes, glycoproteins, and proteins. Affinity capture preprocessing techniques such as combinatorial peptide ligand libraries and biomarker harvesting hydrogel nanoparticles are enabling measurement/identification of previously undetectable urinary proteins. Future challenges in the urinary proteomics field include a) defining either single or multiple, universally applicable data normalization methods for comparing results within and between individual patients/data sets, and b) defining expected urinary protein levels in healthy individuals. disease profile, coupled to our knowledge of biomarkers and prognostic factors [46, 47] . Urinary proteomics is a perfect example of the embodiment of precision medicine in which research is enhancing our assessment of disease risk, elucidating disease mechanisms, and predicting optimal therapy [48] . Extensive reviews of the translational research progress in the urinary proteomic field have been published [2, 41, [49] [50] [51] . Potential urinary biomarkers are being discovered in a variety of non-kidney associated diseases including acute appendicitis [52, 53] , infectious diseases such as Tuberculosis [54], Chagas Disease and Lyme Disease [55-57], cancer [49, 50, 58-61], cardiovascular disease [62-64], and aging [65, 66] . Selected examples of the progress in urinary protein identification are described below, highlighting a) improvements in mass spectrometry-based urinary proteomic discovery, b) the number of identifiable proteins, and c) pathophysiologic associations over the last decade. In 2001, Sphar et. al. established the efficacy of urine protein identification [16]. 124 proteins/expressed sequence tags were identified from unfractionated, pooled normal male urine by applying liquid chromatography coupled mass spectrometry (LC-MS) with iterative peptide ion analysis on a hybrid quadrupole-time-of-flight (Q-TOF) instrument. The combined abundance of 115 proteins represented less than 10% of the sample by mass, thus highlighting the magnitude of low abundance proteins in urine [16]. By 2006, linear ion trap-Fourier transform (LTQ-FT) and linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometers were being utilized for urine protein discovery due to the instruments' high resolution, mass accuracy, wide dynamic range, and fast cycle times. Adachi et. al. identified 1543 urinary proteins in 10 specimens from healthy individuals (one individual and a pool of 9 specimens) [67]. This study identified plasma membrane and lysosomal proteins in the urine, some of which are known to be associated with urinary exosomes, thus underscoring the complex source of urine proteins/peptides [68, 69]. Using high resolution Fourier transform mass spectrometry, Marimuthu et. al. discovered 1823 urine proteins, 671 of which were previously unreported in the urine, from a urine pool specimen comprising 24 healthy individuals [70] . A unique aspect of this study was lectin affinity chromatography with concanavalin A, wheat germ agglutinin and jacalin, to enrich glycoprotein constituents. A comprehensive kidney, urine, and plasma proteome comparison was conducted by Farrah et. al. in 2013 [42]. By combining kidney, urine, and plasma datasets collected from different laboratories, with an estimate of relative protein abundance based on spectral counting and a normalization strategy to compare the proteomes, they were able to identify 2491 nonredundant proteins in the PeptideAtlas (www.peptideatlas.org/hupo/hkup) (Table 2) [71]. Advancements in mass spectrometry based urinary proteomics The ever-increasing depth and breadth of the urinary proteome is attributable to the versatility of mass spectrometry for protein discovery, identification, relative and absolute quantification. Top-down protein profiling begins with a statistically significant number of complex biological samples which are separated by chromatography or 2-D gel electrophoresis. Specific fractions or gel spots containing proteins are analyzed in a mass Harpole et al.
doi:10.1080/14789450.2016.1190651 pmid:27232439 pmcid:PMC4978532 fatcat:3h6zhzwgvfcdvp4ynsgpwhuqpy