Abstract
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, H(2)N-(His)(6)-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO(2)H, which consists of four functional elements: i) an affinity ligand (His(6)-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) Ala-9 residue that contains four (d(4)) or no (d(0)) deuterium atoms, and iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d(4)) or (d(0)) 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-d(4) co-elute with their d(0)-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 Ni(2+) 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 (13)C isotope label, and allows robust purification and flexibility through the affinity tag, which can be extended to different peptide functionalities.
Highlights
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 derivatized decapeptide, H2N-(His)6-Ala-Arg-AlaCys(-2-pyridyl disulfide)-CO2H, which consists of four functional elements: i) an affinity ligand (His6-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) an Ala-9 residue that contains four (d4) or zero (d0) deuterium atoms, and iv) a thiol-reactive group (DPDS)
SDS can be successfully removed from samples by cation exchange chromatography in the presence of moderate concentration of organic solvent [20], but we have found that even successful depletion of the detergent and efficient digestion still leads to difficult-to-analyze samples
Summary
Solid-phase Synthesis of Isotope-labeled HysTag Peptides—The 10-mer HysTag peptides, light and heavy forms, were made by automated solid-phase peptide synthesis on a prototype Peptide Synthesizer (Intavis Bioanalytical Instruments, Cologne, Germany). The spin columns were placed in 2 ml Eppendorf (Hamburg, Germany) tubes and centrifuged at 700 ϫ g for 1 min, and the resin was washed twice with 500 l 25 mM MES, 0.1 M NaCl, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), pH 5.5, followed by 2ϫ wash with 25 mM MES, 0.1 M NaCl, pH 5.5. After 1 h incubation at room temperature, the spin columns were placed in 2 ml Eppendorf tubes and centrifuged at 700 ϫ g for 1 min and the resin was washed three times with 1 ml of B-PER washing buffer supplemented with 1% CHAPS to remove nonspecific-binding hydrophobic peptides. The rather large mass tolerance in MS mode was used to ensure identification of peptide ions selected and isolated by their 13C isotope instead of the 12C isotope via the Analyst software
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