One-Step Sample Concentration, Purification, and Albumin Depletion Method for Urinary Proteomics
TLDR
A one-step sample preparation workflow that simultaneously concentrates proteins, purifies by removing salts and other low molecular weight compounds, and depletes (albumin) from urine samples is developed that can be multiplexed and compatible with a diverse range of downstream multidimensional separation technologies.Abstract:
Workflows in urinary proteomics studies are often complex and require many steps to enrich, purify, deplete, and separate the complex mixture. Many of these methods are laborious, are time-consuming, and have the potential for error. Although individual steps of these methods have been previously studied, their downstream compatibilities with fractionation technologies such as off-gel electrophoresis have not been investigated. We developed a one-step sample preparation workflow that simultaneously (i) concentrates proteins, (ii) purifies by removing salts and other low molecular weight compounds, and (iii) depletes (albumin) from urine samples. This simple and robust workflow can be multiplexed and is compatible with a diverse range of downstream multidimensional separation technologies. Additionally, because of its high reproducibility and flexibility in processing samples with different volumes and concentrations, it has the potential to be used for standardization of urinary proteomics studies, as well as for studying other body fluids of similar complexity.read more
Citations
More filters
Journal ArticleDOI
Effect of quality characteristics of single sample preparation steps in the precision and coverage of proteomic studies--a review.
TL;DR: All sample preparation approaches that maximize yields (percent recoveries) and minimize overall imprecision should be suitable to improve reliability of biomarker search to improve reproducibility and information harvest of a propagated method series.
Journal ArticleDOI
Urinary Proteomics Yield Pathological Insights for Ureteropelvic Junction Obstruction
John W. Froehlich,Stephen A. Kostel,Patricia S. Cho,Andrew C. Briscoe,Hanno Steen,Ali R. Vaezzadeh,Richard S. Lee +6 more
TL;DR: This study gives a deeper understanding of the critical proteomic changes associated with renal obstruction and represents the deepest proteomic profile of renal obstruction to date.
Journal ArticleDOI
Characterizing Patients with Recurrent Urinary Tract Infections in Vesicoureteral Reflux: A Pilot Study of the Urinary Proteome.
Dijana Vitko,Patricia S. Cho,Patricia S. Cho,Stephen A. Kostel,Shannon E. DiMartino,Lily D. Cabour,Matthew A. Migliozzi,Tanya Logvinenko,Peter G. Warren,John W. Froehlich,Richard S. Lee +10 more
TL;DR: Urinary proteomes of children with recurrent infections and vesicoureteral reflux show differential expression of proteins involved in inflammation, acute phase response (APR), modulation of extracellular matrix (ECM), and carbohydrate metabolism, which may represent candidate biomarkers to characterize susceptibility of infection and renal damage towards improving medical care for patients.
Journal ArticleDOI
Analytical approaches to assess metabolic changes in psoriasis.
TL;DR: A wide range of techniques involved in lipidomic research based on a combination of mass spectrometry and different types of chromatography have allowed comprehensive assessment of lipid modification in psoriatic skin and provided new insight into the role of lipids and their mechanism of action in psoriasis.
Journal ArticleDOI
A novel approach for the chromatographic purification and peptide mass fingerprinting of urinary free light chains.
TL;DR: This is the first report of characterization and validation of FLCs from large volume samples by peptide sequencing, and this simple and cost-effective approach to purification of F LCs, together with the easy availability of urine samples make the large-scale production of FLP possible, allowing exploration of various bioclinical as well as biodiagnostic applications.
References
More filters
Journal ArticleDOI
Large-scale analysis of the yeast proteome by multidimensional protein identification technology.
TL;DR: MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date, identifying 131 proteins with three or more predicted transmembrane domains which allowed us to map the soluble domains of many of the integral membrane proteins.
Journal ArticleDOI
The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins.
Jun Adachi,Chanchal Kumar,Yanling Zhang,Yanling Zhang,Jesper V. Olsen,Jesper V. Olsen,Matthias Mann +6 more
TL;DR: The analysis provides a high-confidence set of proteins present in human urinary proteome and provides a useful reference for comparing datasets obtained using different methodologies and may prove useful in biomarker discovery in the future.
Journal ArticleDOI
Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography−Tandem Mass Spectrometry
David L. Tabb,Lorenzo Vega-Montoto,Lorenzo Vega-Montoto,Paul A. Rudnick,Asokan Mulayath Variyath,Asokan Mulayath Variyath,Amy-Joan L. Ham,David M. Bunk,Lisa E. Kilpatrick,Dean Billheimer,Ronald K. Blackman,Helene L. Cardasis,Helene L. Cardasis,Steven A. Carr,Karl R. Clauser,Jacob D. Jaffe,Kevin A. Kowalski,Thomas A. Neubert,Fred E. Regnier,Birgit Schilling,Tony J. Tegeler,Mu Wang,Pei Wang,Jeffrey R. Whiteaker,Lisa J. Zimmerman,Susan J. Fisher,Bradford W. Gibson,Christopher R. Kinsinger,Mehdi Mesri,Henry Rodriguez,Stephen E. Stein,Paul Tempst,Amanda G. Paulovich,Daniel C. Liebler,Cliff Spiegelman +34 more
TL;DR: The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides.
Journal ArticleDOI
Characterization of the human urinary proteome: A method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots
Rembert Pieper,Christine L. Gatlin,Andrew M. McGrath,Anthony J. Makusky,Madhu Mondal,Michael D Seonarain,Erin Field,Courtney R. Schatz,Marla A. Estock,Nasir Ahmed,Norman G. Anderson,Sandra Steiner +11 more
TL;DR: A protein fractionation strategy enriching proteins of molecular masses lower than 30 kDa in a fraction separate from larger proteins is described, which led to the successful identification of 30% of the proteins.
Journal ArticleDOI
Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest.
Chris Spahr,Michael T. Davis,Michael D. McGinley,John H. Robinson,Edward J. Bures,Jill Beierle,Jessica Mort,Paul L. Courchesne,Kui Chen,Robert C. Wahl,Wen Yu,Roland Luethy,Scott D. Patterson +12 more
TL;DR: The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography‐tandem mass spectrometry (LC‐MS/MS), and the experimental time for these analyses was less than that required to run a single two‐dimensional gel.