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Journal ArticleDOI

One-Step Sample Concentration, Purification, and Albumin Depletion Method for Urinary Proteomics

05 Nov 2010-Journal of Proteome Research (J Proteome Res)-Vol. 9, Iss: 11, pp 6082-6089
TL;DR: 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.

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Citations
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Journal ArticleDOI
TL;DR: In this paper, an in-depth analysis of the urinary proteome is presented based on different separation strategies, including direct one dimensional liquid chromatography–tandem mass spectrometry (LC/MS/MS), two dimensional LC/ MS/MS, and gel-eluted liquid fraction entrapment electrophoresis/liquid-phase isoelectric focusing followed by two dimensional Liquid Chromatography-MS-MS.
Abstract: Biomarkers are measurable changes associated with the disease Urine can reflect the changes of the body while blood is under control of the homeostatic mechanisms; thus, urine is considered an important source for early and sensitive disease biomarker discovery A comprehensive profile of the urinary proteome will provide a basic understanding of urinary proteins In this paper, we present an in-depth analysis of the urinary proteome based on different separation strategies, including direct one dimensional liquid chromatography–tandem mass spectrometry (LC/MS/MS), two dimensional LC/MS/MS, and gel-eluted liquid fraction entrapment electrophoresis/liquid-phase isoelectric focusing followed by two dimensional LC/MS/MS A total of 6085 proteins were identified in healthy urine, of which 2001 were not reported in previous studies and the concentrations of 2571 proteins were estimated (spanning a magnitude of 106) with an intensity-based absolute quantification algorithm The urinary proteins were annotated by their tissue distribution Detailed information can be accessed at the “Human Urine Proteome Database” (wwwurimarkercom/urine)

122 citations

Journal ArticleDOI
TL;DR: The aim of this review article is to give a concise report on the latest developments in the field of proteomic sample preparation by expanding on capturing/enriching subglycoproteomics by lectin affinity chromatography.
Abstract: This review article expands on the previous one (S. Selvaraju and Z. El Rassi, Electrophoresis 2012, 33, 74-88) by reviewing pertinent literature in the period extending from early 2011 to present. As the previous review article, the present one is concerned with proteomic sample preparation (e.g., depletion of high abundance proteins, reduction of the protein dynamic concentration range, enrichment of a particular sub-proteome), and the subsequent chromatographic and/or electrophoretic pre-fractionation prior to peptide separation and identification by LC-MS/MS. This review article is distinguished from its second version published in Electrophoresis 2012, 33, 74-88 by expanding on capturing/enriching sub-phosphoproteomes by immobilized metal affinity chromatography and metal oxide affinity chromatography. Seventy-seven papers published in the period extending from mid 2011 to the present have been reviewed. By no means this review article is exhaustive, given the fact that its aim is to give a concise treatment of the latest developments in the field.

84 citations

Journal ArticleDOI
TL;DR: It is clear that the field of proteomics has not yet fulfilled its promise, but ongoing efforts to standardize sample collection and preparation, improve study designs, perform multicenter validations, and create joint industry-regulatory bodies offer promise for the recognition of novel molecules that could change clinical nephrology forever.
Abstract: BACKGROUND: Technological advances have resulted in a renaissance of proteomic studies directed at finding markers of disease progression, diagnosis, or responsiveness to therapy. Renal diseases are ideally suited for such research, given that urine is an easily accessible biofluid and its protein content is derived mainly from the kidney. Current renal prognostic markers have limited value, and renal biopsy remains the sole method for establishing a diagnosis. Mass spectrometry instruments, which can detect thousands of proteins at nanomolar (or even femtomolar) concentrations, may be expected to allow the discovery of improved markers of progression, diagnosis, or treatment responsiveness. CONTENT: In this review we describe the strengths and limitations of proteomic methods and the drawbacks of existing biomarkers, and provide an overview of opportunities in the field. We also highlight several proteomic studies of biomarkers of renal diseases selected from the plethora of studies performed. SUMMARY: It is clear that the field of proteomics has not yet fulfilled its promise. However, ongoing efforts to standardize sample collection and preparation, improve study designs, perform multicenter validations, and create joint industry–regulatory bodies offer promise for the recognition of novel molecules that could change clinical nephrology forever.

40 citations

Journal ArticleDOI
TL;DR: A simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of N- glycans, N-glycosites, and proteins in a single workflow.

33 citations


Cites background or methods from "One-Step Sample Concentration, Puri..."

  • ...Isoelectric Focusing of Peptides—The tryptic peptides from all urine and plasma samples were focused into 24 fractions using a 3100 OFFGEL fractionator (Agilent, Santa Clara, CA) as described previously (17)....

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  • ...To demonstrate its advantage for glycomics research, urine was tested because it is a complex protein analyte (34), which contains significant amounts of chemically heterogeneous metabolites and salts (17)....

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  • ...The processing of urine samples and the depletion of albumin in urine were performed according to the reported one-step protocol (17)....

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Journal ArticleDOI
TL;DR: This review describes the current state of isoelectric focusing (IEF) gel-free electrophoresis based on the Agilent offgel 3100 fractionator and discusses offgel fundamentals and future trends.

31 citations

References
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Journal ArticleDOI
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.
Abstract: We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). 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. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.

4,805 citations

Journal ArticleDOI
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.
Abstract: Urine is a desirable material for the diagnosis and classification of diseases because of the convenience of its collection in large amounts; however, all of the urinary proteome catalogs currently being generated have limitations in their depth and confidence of identification. Our laboratory has developed methods for the in-depth characterization of body fluids; these involve a linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometer. Here we applied these methods to the analysis of the human urinary proteome. We employed one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography for protein separation and fractionation. Fractionated proteins were digested in-gel or in-solution, and digests were analyzed with the LTQ-FT and LTQ-Orbitrap at parts per million accuracy and with two consecutive stages of mass spectrometric fragmentation. We identified 1543 proteins in urine obtained from ten healthy donors, while essentially eliminating false-positive identifications. Surprisingly, nearly half of the annotated proteins were membrane proteins according to Gene Ontology (GO) analysis. Furthermore, extracellular, lysosomal, and plasma membrane proteins were enriched in the urine compared with all GO entries. Plasma membrane proteins are probably present in urine by secretion in exosomes. Our 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. The urinary proteome is unexpectedly complex and may prove useful in biomarker discovery in the future.

647 citations

Journal ArticleDOI
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.
Abstract: The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproduci...

504 citations

Journal ArticleDOI
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.
Abstract: The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (Mr) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with Mrs higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.

345 citations

Journal ArticleDOI
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.
Abstract: The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography-tandem mass spectrometry (LC-MS/MS). The entire urinary protein mixture was denatured, reduced and enzymatically digested prior to LC-MS/MS analysis using a hybrid-quadrupole time-of-flight mass spectrometer (Q-TOF) to perform data-dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture was analyzed four separate times, with the mass spectrometer selecting ions for fragmentation from a subset of the entire mass range for each run. This approach requires only an autosampler on the HPLC for automation ( i.e, unattended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and translations of expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a single two-dimensional gel.

296 citations