Searching across hundreds of databases
PeptideAtlas provides a multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments collected for human, mouse, yeast, and several other organisms.
(last updated: Nov 28, 2017)
Data or Model Molecule| Accession | Sample Title | Summary | Organism | Characteristics | Instrument | Treatment | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| PAe000225 | youngah_yagwall_712rpt | enrichment for Halobacterium cell wall proteins; 700-1200 m/z; Goo et al., unpublished data. Young Ah Goo ICAT experiments - yagwall_712rpt directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000205 | youngah_yagwall_48rpt | enrichment for Halobacterium cell wall proteins; 400-800m/z; Goo et al., unpublished data. Young Ah Goo ICAT experiments - yagwall_48rpt directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000254 | youngah_yagwall_1120rpt | enrichment for Halobacterium cell wall proteins; 1100-2000 m/z; Goo et al., unpublished data. Young Ah Goo ICAT experiments - yagwall_1120rpt directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000209 | youngah_YAGwall | enrichment for Halobacterium cell wall proteins, Goo et al., unpublished data. Young Ah Goo ICAT experiments - YAGwall directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000241 | youngah_yagsn2_712rpt | replicate enrichment for soluble proteins; 700-1200 m/z; Goo et al., 2003, Mol Cell Proteomics 2(8):512 Young Ah Goo ICAT experiments - yagsn2_712rpt directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000218 | youngah_YAGsn2_48rpt | replicate enrichment for soluble proteins; 400-800 m/z; Goo et al., 2003, Mol Cell Proteomics 2(8):511 Young Ah Goo ICAT experiments - YAGsn2_48rpt directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000181 | youngah_yagsn2_1120rpt | replicate enrichment for soluble proteins; 1100-2000 m/z; Goo et al., 2003, Mol Cell Proteomics 2(8):510 Young Ah Goo ICAT experiments - yagsn2_1120rpt directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000249 | youngah_YAGsn2 | replicate enrichment for soluble proteins; Goo et al., 2003, Mol Cell Proteomics 2(8):509 Young Ah Goo ICAT experiments - YAGsn2 directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000210 | youngah_yagsn1_712rpt | replicate enrichment for soluble proteins; 700-1200 m/z; Goo et al., 2003, Mol Cell Proteomics 2(8):508 Young Ah Goo ICAT experiments - yagsn1_712rpt directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000220 | youngah_yagsn1_48rpt | replicate enrichment for soluble proteins; 400-800 m/z; Goo et al., 2003, Mol Cell Proteomics 2(8):507 Young Ah Goo ICAT experiments - yagsn1_48rpt directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000173 | youngah_yagsn1_1120rpt | replicate enrichment for soluble proteins; 1100-2000 m/z; Goo et al., 2003, Mol Cell Proteomics 2(8):506 Young Ah Goo ICAT experiments - yagsn1_1120rpt directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000224 | youngah_YAGsn1 | enrichment for soluble proteins; Goo et al., 2003, Mol Cell Proteomics 2(8):505 Young Ah Goo ICAT experiments - YAGsn1 directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000240 | youngah_yagpt | enrichment for proteins in precipitant following ultracentrifugation; Goo et al., 2003, Mol Cell Proteomics 2(8):505 Young Ah Goo ICAT experiments - yagpt directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000189 | youngah_yagpm3_3 | enrichment for specialized purple membrane proteins, cell culture #3; Goo et al., unpublished data. Young Ah Goo ICAT experiments - yagpm3_3 directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000187 | youngah_yagpm3_2 | enrichment for specialized purple membrane proteins, cell culture #3; Goo et al., unpublished data. Young Ah Goo ICAT experiments - yagpm3_2 directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000193 | youngah_yagpm3_1 | enrichment for specialized purple membrane proteins, cell culture #3; Goo et al., unpublished data. Young Ah Goo ICAT experiments - yagpm3_1 directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000203 | youngah_yagpm2_3 | enrichment for specialized purple membrane proteins, cell culture #2 Goo et al., unpublished data. Young Ah Goo ICAT experiments - yagpm2_3 directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000252 | youngah_yagpm2_2 | enrichment for specialized purple membrane proteins, cell culture #2 Goo et al., unpublished data. Young Ah Goo ICAT experiments - yagpm2_2 directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000256 | youngah_yagpm2_1 | enrichment for specialized purple membrane proteins, cell culture #2 Goo et al., unpublished data. Young Ah Goo ICAT experiments - yagpm2_1 directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 | ||
| PAe000247 | youngah_yagpm | enrichment for specialized purple membrane proteins, cell culture #1, Goo et al., unpublished data. Young Ah Goo ICAT experiments - yagpm directory | Halobacterium | Halobacterium sp. NRC-1 (ATCC700922) | LCQ DECA | Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678. | One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis. | Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. | PMID:12872007 |
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