Exploring the Hidden Human Urinary Proteome via Ligand Library Beads

Castagna, Annalisa; Cecconi, Daniela; Sennels, Lau; Rappsilber, Juri; Guerrier, Luc; Fortis, Fréderic; Boschetti, Egisto; Lomas, Lee; Righetti, Pier Giorgio

doi:10.1021/pr050153r

Cited by 230 publications

(179 citation statements)

References 40 publications

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“…As described, about 7% of abundant urine proteins (Castagna et al, 2005), about 5% of red blood cell proteins, and even 13% of platelets' extracted proteins, well detected before the treatment, are not detectable any longer in the eluted proteins from the beads. This situation suggests that either some proteins do not find a peptide partner to form a stable complex, or the protein-peptide complex is governed by a strong association constant preventing efficient recovery with current eluting agents.…”

Section: A the General Behavior Of Hexapeptide Librariesmentioning

confidence: 60%

“…Its application to proteomics, though, is relatively recent, because the first report appeared only 3 years ago (Thulasiraman et al, 2005). A flurry of applications soon followed; for example, in urine (Castagna et al, 2005), serum Sennels et al, 2007), human platelets (Guerrier et al, 2007a), red blood cell (Roux-Dalvai et al, 2008), bile (Guerrier et al, 2007b), and recombinant DNA product Antonioli et al, 2007) analyses. This technology is presently commercially available under the trade name of ProteoMiner.…”

Section: Introductionmentioning

confidence: 99%

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The ProteoMiner and the FortyNiners: Searching for gold nuggets in the proteomic arena

Righetti

Boschetti

2008

Mass Spectrometry Reviews

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128

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The present review covers modern aspects of combinatorial peptide ligand libraries (CPLL), as used to analyze the ''lowabundance proteome'' in association with mass spectrometry. First, the capturing properties of baits of different lengths (from single amino acid to hexa-peptides) are described to show that a plateau is rapidly reached above a tetra-peptide in length, thus confirming the validity of having adopted hexapeptides for the considered application. The mechanism of interaction with proteins from very complex proteomes and the ability to decrease the dynamic concentration range is demonstrated with the help of mass spectrometry analysis. Examples are given on how treatment with CPLLs dramatically improves the detectability of peptides in mass spectrometry analysis, permitting detection of a very large number of proteins as compared with control, untreated samples. The use of complementary libraries is discussed with the aim to discover additional low-abundance species that escaped the first library. A discussion on the possibility to discover extremely rare gene products, and the quantitative aspect of the technology when associated with mass spectrometry is also provided. Some insights on the applications for hidden, low-abundance biomarkers are also presented. #

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Section: A the General Behavior Of Hexapeptide Librariesmentioning

confidence: 60%

Section: Introductionmentioning

confidence: 99%

The ProteoMiner and the FortyNiners: Searching for gold nuggets in the proteomic arena

Righetti

Boschetti

2008

Mass Spectrometry Reviews

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128

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“…For combinatorial peptide ligand libraries, we referred to a paper for human urinary proteins analysis. 39 The results showed the number of proteins from a treated human urine sample was improved 186% compared with that from an untreated one. For scFv@M13@MM, the number of proteins from the treated human serum sample was improved 100% compared with that from the untreated one.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Single Chain Variable Fragment Displaying M13 Phage Library Functionalized Magnetic Microsphere-Based Protein Equalizer for Human Serum Protein Analysis

et al. 2012

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Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@ M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (GlyHCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC−ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@ M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification. S earching indicators or biomarkers for diseases is one of the most important issues in clinical proteomics. Among various body fluids, serum is the one of the most favorite clinical samples, because not only it contains most possible biomarkers of diseases, 1,2 but also the collection is minimally invasive.However, the enormous dynamic range of protein concentration makes serum the most difficult specimen to be dealt with by existing techniques. The few dozen of highabundance proteins make up 99% of overall contents, which seriously undermine the identification of low-abundance ones. 3−5 Therefore, the decrease of the dynamic range of protein concentration in serum is essential to discover lowabundance ones. Until now, there are two main strategies to decrease the dynamic range of protein concentration in serum, depletion of high-abundance proteins, 6−14 and equalization of protein abundance. 15−28 For depletion of high-abundance proteins in serum samples, several kinds of immnoaffinity columns have been widely used. 6−13 Recently, Zeng et al. 10 applied CaptureSelect depletion resins (from BAC. B.V.) to remove albumin and immunoglobulin from human serum, followed by highperformance multilectin affinity chromatography fractionation, isoelectric focusing fractionation, in-gel digestion, and reversedplase liquid chromatography−electrospray ionization-tandem mass sp...

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“…It is realized however, that the consequence of discarding the sediments may vary depending on the analytical protocol. For example, using the ligand library bead method, as described by Castagna et al [24], less emphasis is placed on protein concentration, and thus discarding the sediments may not significantly alter the resulting protein profile. However, discarding the sediments while using this method may still impose consequences with respect to unique sediment proteins since these would ultimately be discarded.…”

Section: Analysis Of the Sediment Proteome Has Implications For Biomamentioning

confidence: 99%

A qualitative proteome investigation of the sediment portion of human urine: Implications in the biomarker discovery process

Mataija-Botelho¹,

Murphy²,

Pinto³

et al. 2009

Proteomics Clinical Apps

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Inherent to the biomarker discovery process is a comparative analysis of physiological states. It is therefore critical that the proteome detection protocol does not bias the analysis. With urine, the sediment portion, obtained upon thawing frozen urine, is routinely discarded prior to proteome analysis. However, our results demonstrate that such a practice inadvertently induces bias, having significant implications in the biomarker discovery process. We present the first proteome investigation of human urinary sediments, identifying 60 proteins in this phase by MS. Many sediment proteins were also detected in the urinary supernatant, indicating that several proteins partition between the two phases. This partitioning is dependant on the pH of the sample, as well as the degree of sample agitation. As a consequence of discarding the sediment portion of urine, the concentration of potential candidate biomarkers in the supernatant phase will be altered or, in other instances, may be completely removed from the sample. To minimize this, the pH of all samples should first be normalized, and the samples vigorously vortexed prior to discarding the sediments. For more comprehensive biomarker investigations, we suggest that urinary sediments be analyzed along with the supernatant proteins.

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Exploring the Hidden Human Urinary Proteome via Ligand Library Beads

Cited by 230 publications

References 40 publications

The ProteoMiner and the FortyNiners: Searching for gold nuggets in the proteomic arena

The ProteoMiner and the FortyNiners: Searching for gold nuggets in the proteomic arena

Single Chain Variable Fragment Displaying M13 Phage Library Functionalized Magnetic Microsphere-Based Protein Equalizer for Human Serum Protein Analysis

A qualitative proteome investigation of the sediment portion of human urine: Implications in the biomarker discovery process

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