HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain

Research output: Contribution to journalJournal articlepeer-review

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HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain. / Olsen, Jesper Velgaard; Andersen, Jens Roswalld; Nielsen, Peter Aa; Nielsen, Michael Lund; Figeys, Daniel; Mann, Matthias; Wisniewski, Jacek R.

In: Molecular and Cellular Proteomics, Vol. 3, No. 1, 01.2004, p. 82-92.

Research output: Contribution to journalJournal articlepeer-review

Harvard

Olsen, JV, Andersen, JR, Nielsen, PA, Nielsen, ML, Figeys, D, Mann, M & Wisniewski, JR 2004, 'HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain', Molecular and Cellular Proteomics, vol. 3, no. 1, pp. 82-92. https://doi.org/10.1074/mcp.M300103-MCP200

APA

Olsen, J. V., Andersen, J. R., Nielsen, P. A., Nielsen, M. L., Figeys, D., Mann, M., & Wisniewski, J. R. (2004). HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain. Molecular and Cellular Proteomics, 3(1), 82-92. https://doi.org/10.1074/mcp.M300103-MCP200

Vancouver

Olsen JV, Andersen JR, Nielsen PA, Nielsen ML, Figeys D, Mann M et al. HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain. Molecular and Cellular Proteomics. 2004 Jan;3(1):82-92. https://doi.org/10.1074/mcp.M300103-MCP200

Author

Olsen, Jesper Velgaard ; Andersen, Jens Roswalld ; Nielsen, Peter Aa ; Nielsen, Michael Lund ; Figeys, Daniel ; Mann, Matthias ; Wisniewski, Jacek R. / HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain. In: Molecular and Cellular Proteomics. 2004 ; Vol. 3, No. 1. pp. 82-92.

Bibtex

@article{308ce8c0e97511deba73000ea68e967b,
title = "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 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.",
author = "Olsen, {Jesper Velgaard} and Andersen, {Jens Roswalld} and Nielsen, {Peter Aa} and Nielsen, {Michael Lund} and Daniel Figeys and Matthias Mann and Wisniewski, {Jacek R}",
note = "Keywords: Amino Acid Sequence; Animals; Brain Chemistry; Cysteine; Gene Expression Profiling; Indicators and Reagents; Membrane Proteins; Mice; Molecular Structure; Peptide Fragments; Proteomics",
year = "2004",
month = jan,
doi = "10.1074/mcp.M300103-MCP200",
language = "English",
volume = "3",
pages = "82--92",
journal = "Molecular and Cellular Proteomics",
issn = "1535-9476",
publisher = "American Society for Biochemistry and Molecular Biology",
number = "1",

}

RIS

TY - JOUR

T1 - HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain

AU - Olsen, Jesper Velgaard

AU - Andersen, Jens Roswalld

AU - Nielsen, Peter Aa

AU - Nielsen, Michael Lund

AU - Figeys, Daniel

AU - Mann, Matthias

AU - Wisniewski, Jacek R

N1 - Keywords: Amino Acid Sequence; Animals; Brain Chemistry; Cysteine; Gene Expression Profiling; Indicators and Reagents; Membrane Proteins; Mice; Molecular Structure; Peptide Fragments; Proteomics

PY - 2004/1

Y1 - 2004/1

N2 - 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.

AB - 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.

U2 - 10.1074/mcp.M300103-MCP200

DO - 10.1074/mcp.M300103-MCP200

M3 - Journal article

C2 - 14610161

VL - 3

SP - 82

EP - 92

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 1

ER -

ID: 16275875