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

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 chargebased 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 cationexchange 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.Molecular & Cellular Proteomics 3: 82-92, 2004.

Proteomic Mapping of Brain Plasma Membrane Proteins

“…We have previously employed a classical fractionation approach for quantitative mapping of proteins in mouse fore-and hindbrain with a total of 300 mg of tissue (10). Except cortex, all other mouse brain compartments are smaller than 100 mg; therefore, to map proteins in distinct areas of mouse brain and to avoid combining tissues from several animals, development of an alternative approach is required.…”

“…Advantages of digestion of integral membrane proteins of intact membranes have been demonstrated recently (10,27). The membrane can be considered as a solid phase carrying polypeptides that can be modified while immobilized.…”

“…Disulphide bond reduction, thiol alkylation, and digestion of the polypeptides is facilitated in this way. Recently, we have demonstrated that this procedure can be applied to modification of thiol-containing peptides for complexity reduction and relative quantification of membrane proteins by the HysTag reagent (10).…”

“…In this study, we focused on one of the most valuable cellular compartment for molecular biology and drug discovery-the PM. Our approach combines a new PM purification method, digestion of membrane proteins on nonsolubilized membranes (10,27), diagonal chromatography, and highly sensitive MS. The novel method for enrichment of PMs from frozen tissues has been applied to mapping of cortical and hippocampal membrane proteins.…”

“…Yoshimura (8) identified 492 proteins using LC-MS/MS, whereas the two other approaches identified far fewer proteins. Recently, application of LC-MS/MS for comparative analysis of fractions enriched in plasma membranes (PM) from mouse fore-and hindbrain resulted in identification of 355 and relative quantification of 281 proteins (10).…”