Archived Hot Topics
Below you will be able to find all the Hot Topic's
that were once posted on the PCL main page.
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Nature Biotechnology
23
Enrichment and Analysis of Peptide Subsets Using Fluorous Affinity Tags and Mass Spectrometry
Although mass spectrometry has become a powerful tool for the functional analysis of biological systems, complete proteome characterization cannot yet be achieved. Instead, the sheer complexity of living organisms demands fractionation of cellular extracts to enable more targeted analyses. Here, we introduce the concept of 'fluorous proteomics,' whereby specific peptide subsets from samples of biological origin are tagged with perfluorinated moieties and subsequently enriched by solid-phase extraction over a fluorous-functionalized stationary phase. This approach is extremely selective, yet can readily be tailored to enrich different subsets of peptides. Additionally, this methodology overcomes many of the limitations of traditional bioaffinity-based enrichment strategies, while enabling new affinity enrichment schemes impossible to implement with bioaffinity reagents. The potential of this methodology is demonstrated by the facile enrichment of peptides bearing particular side-chain functionalities or post-translational modifications from tryptic digests of individual proteins as well as whole cell lysates.
Click here to read more.
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Nature Biotechnology
December 2003; Vol 21, pp 1509-1512
Analyzing antibody specificity with whole proteome microarrays
Although approximately 10,000 antibodies are available from commercial sources, antibody reagents are still unavailable for most proteins1. Furthermore, new applications such as antibody arrays2-5 and monoclonal antibody therapeutics6, 7 have increased the demand for more specific antibodies to reduce cross-reactivity and side effects. An array containing every protein for the relevant organism represents the ideal format for an assay to test antibody specificity, because it allows the simultaneous screening of thousands of proteins for possible cross-reactivity. As an initial test of this approach, we screened 11 polyclonal and monoclonal antibodies to 5,000 different yeast proteins deposited on a glass slide and found that, in addition to recognizing their cognate proteins, the antibodies cross-reacted with other yeast proteins to varying degrees. Some of the interactions of the antibodies with noncognate proteins could be deduced by alignment of the primary amino acid sequences of the antigens and cross-reactive proteins; however, these interactions could not be predicted a priori. Our findings show that proteome array technology has potential to improve antibody design and selection for applications in both medicine and research.
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Nature Biotechnology
Vol. 21, No. 9 (17 August 2003)
Phosphospecific proteolysis for mapping sites of protein phosphorylation. Knight, Z.A., et al
Protein phosphorylation is a dominant mechanism of information transfer in cells, and a major goal of current proteomic efforts is to generate a system-level map describing all the sites of protein phosphorylation. Recent efforts have focused on developing technologies for enriching and quantifying phosphopeptides. Identification of the sites of phosphorylation typically relies on tandem mass spectrometry to sequence individual peptides. Here we describe an approach for phosphopeptide mapping that makes it possible to interrogate a protein sequence directly with a protease that recognizes sites of phosphorylation. The key to this approach is the selective chemical transformation of phosphoserine and phosphothreonine residues into lysine analogs (aminoethylcysteine and -methylaminoethylcysteine, respectively). Aminoethylcysteine-modified peptides are then cleaved with a lysine-specific protease to map sites of phosphorylation. A blocking step enables single-site cleavage, and adaptation of this reaction to the solid phase facilitates phosphopeptide enrichment and modification in one step Click here to read more.
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Nature Biotechnology
Vol. 21, No. 6 (18 May 2003).
Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins Kaji, H. et al
The authors describe a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, 18O, specifically into the N-glycosylation site. The 18O-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. Click here to read more.
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Another Publication of note:
Proteomics in 2002: A year of technical development and wide-ranging applications. Figeys, D. Anal. Chem. 2003, 75, 2891-2905
Over 1300 papers with the word proteome or proteomics(s) were reported by PubMed for 2002. This paper presents an interesting review of about 200 publications from 2002 that illustrate the breadth of proteomics development and applications during the past year.
Included is a paper by TAMU Research Scientist Bill Russell (Department of Chemistry), demonstrating that the rate of protein digestion using trypsin can be improved using digestion conditions that incorporate organic solvent. This is useful, particularly for membrane proteins. Check it out (Proteolysis in Mixed Organic-Aqueous Solvent Systems: Applications for Peptide Mass Mapping Using Mass Spectrometry. Russell, W. et al 2001 Anal. Chem. 73 (5), 2682-2685 Click here to read more.
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Nature Biotechnology
Vol. 21, No. 5 (14 April 2003).
A method for the comprehensive proteomic analysis of membrane proteins. C.C. Wu, M.J. MacCoss, K.E. Howell and J.R. Yates, III
A new method (hpPK; high pH and proteinase K) developed by John Yates’ group allows for the concurrent proteomic analysis of membrane and soluble proteins from complex membrane-containing samples. When coupled with MudPIT technology, this method results in the identification of soluble and membrane proteins, (ii) the identification of post-translational modification sites and (iii) the characterization of membrane protein topology and relative localization of soluble proteins. Click here to read more.
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Proteomics
Vol. 422, No. 6928 (13 March 2003)
The term proteome defines the entire protein complement in a given cell, tissue or organism. In its wider sense, proteomics research also assesses protein activities, modifications and localization, and interactions of proteins in complexes. Proteomics promises to transform biological and medical research, and this Insight reviews the progress made in this technology-driven enterprise and looks at future challenges and both basic and clinical applications. Click here to read more.
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