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Molecular & Cellular Proteomics 3:82-92, 2004.
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

HysTag—A Novel Proteomic Quantification Tool Applied to Differential Display Analysis of Membrane Proteins From Distinct Areas of Mouse Brain^*

Jesper V. Olsen^,, Jens R. Andersen^,¶, Peter Aa. Nielsen^,¶, Michael L. Nielsen^,||, Daniel Figeys, Matthias Mann^,**, and Jacek R. Winiewski^,¶,

From the MDS Proteomics A/S, Stærmosegårdsvej 6, DK-5230 Odense M, Denmark; and ^** Center for Experimental BioInformatics, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

A novel isotopically labeled cysteine-tagging and complexity-reducing reagent, called HysTag, has been synthesized and used for quantitative proteomics of proteins from enriched plasma membrane preparations from mouse fore- and hindbrain. The reagent is a 10-mer derivatized peptide, H₂N-(His)₆-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO₂H, which consists of four functional elements: i) an affinity ligand (His₆-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) Ala-9 residue that contains four (d₄) or no (d₀) deuterium atoms, and iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d₄) or (d₀) reagent. The HysTag peptide is preserved in Lys-C digestion of proteins and allows charge-based selection of cysteine-containing peptides, whereas subsequent tryptic digestion reduces the labeling group to a di-peptide, which does not hinder effective fragmentation. Furthermore, we found that tagged peptides containing Ala-d₄ co-elute with their d₀-labeled counterparts. To demonstrate effectiveness of the reagent, a differential analysis of mouse forebrain versus hindbrain plasma membranes was performed. Enriched plasma membrane fractions were partially denatured, reduced, and reacted with the reagent. Digestion with endoproteinase Lys-C was carried out on nonsolubilized membranes. The membranes were sedimented by ultra centrifugation, and the tagged peptides were isolated by Ni²⁺ affinity or cation-exchange chromatography. Finally, the tagged peptides were cleaved with trypsin to release the histidine tag (residues 1–8 of the reagent) followed by liquid chromatography tandem mass spectroscopy for relative protein quantification and identification. A total of 355 unique proteins were identified, among which 281 could be quantified. Among a large majority of proteins with ratios close to one, a few proteins with significant quantitative changes were retrieved. The HysTag offers advantages compared with the isotope-coded affinity tag reagent, because the HysTag reagent is easy to synthesize, economical due to use of deuterium instead of ¹³C isotope label, and allows robust purification and flexibility through the affinity tag, which can be extended to different peptide functionalities.

^¶ Current address: MDS Inc., Denmark, Stærmosegårdsvej 6, DK-5230 Odense M, Denmark

^|| Current address: Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, Husargatan 3, Box 583, SE-75 123 Uppsala, Sweden

To whom correspondence should be addressed: Jacek R. Winiewski, MDS Inc. Denmark, Stærmosegårdsvej 6, DK-5230 Odense M, Denmark. Fax: 45-65-57-20-01; E-mail: jwisniewski{at}mdsdenmark.com; or Matthias Mann, Center for Experimental Bioinformatics, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. Fax: 45-65-93-30-18; E-mail: mann{at}bmb.sdu.dk

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