ltq mass spectrometer  (Thermo Fisher)


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    Structured Review

    Thermo Fisher ltq mass spectrometer
    Mass spectrometry analysis of immunopurified protein interactors for QIL1, MIC27, MIC19, MIC25, MIC60, MTX2, DNAJC11 and GFP in 293T and/or HCT116 cells. Spectra search with Sequest, target-decoy peptide filtering, and linear discriminant analysis ( Huttlin et al., 2010 ) was performed on raw data obtained from technical duplicate runs on either an Thermo <t>LTQ</t> or a LTQ <t>Orbitrap</t> Elite mass spectrometer. Protein Assembler was used to convert spectral counts to APSMs. Peptide data (APSMs) were uploaded into the CompPASS algorithm housed within the CORE environment. Interaction confidence was ranked according to the NWD scores ( Sowa et al., 2009 ). The results obtained from the CompPASS analysis were filtered to determine proteins with a NWD score > 1 or high confidence interactors (HCIPs). HCIPs were then filtered for mitochondrial proteins based on the proteins present in Mitocarta ( Pagliarini et al., 2008 ). For the 293T data set, HCIPs with 2 or more ASPMs were included. DOI: http://dx.doi.org/10.7554/eLife.06265.005
    Ltq Mass Spectrometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 643 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ltq mass spectrometer/product/Thermo Fisher
    Average 95 stars, based on 643 article reviews
    Price from $9.99 to $1999.99
    ltq mass spectrometer - by Bioz Stars, 2020-05
    95/100 stars

    Images

    1) Product Images from "QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology"

    Article Title: QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology

    Journal: eLife

    doi: 10.7554/eLife.06265

    Mass spectrometry analysis of immunopurified protein interactors for QIL1, MIC27, MIC19, MIC25, MIC60, MTX2, DNAJC11 and GFP in 293T and/or HCT116 cells. Spectra search with Sequest, target-decoy peptide filtering, and linear discriminant analysis ( Huttlin et al., 2010 ) was performed on raw data obtained from technical duplicate runs on either an Thermo LTQ or a LTQ Orbitrap Elite mass spectrometer. Protein Assembler was used to convert spectral counts to APSMs. Peptide data (APSMs) were uploaded into the CompPASS algorithm housed within the CORE environment. Interaction confidence was ranked according to the NWD scores ( Sowa et al., 2009 ). The results obtained from the CompPASS analysis were filtered to determine proteins with a NWD score > 1 or high confidence interactors (HCIPs). HCIPs were then filtered for mitochondrial proteins based on the proteins present in Mitocarta ( Pagliarini et al., 2008 ). For the 293T data set, HCIPs with 2 or more ASPMs were included. DOI: http://dx.doi.org/10.7554/eLife.06265.005
    Figure Legend Snippet: Mass spectrometry analysis of immunopurified protein interactors for QIL1, MIC27, MIC19, MIC25, MIC60, MTX2, DNAJC11 and GFP in 293T and/or HCT116 cells. Spectra search with Sequest, target-decoy peptide filtering, and linear discriminant analysis ( Huttlin et al., 2010 ) was performed on raw data obtained from technical duplicate runs on either an Thermo LTQ or a LTQ Orbitrap Elite mass spectrometer. Protein Assembler was used to convert spectral counts to APSMs. Peptide data (APSMs) were uploaded into the CompPASS algorithm housed within the CORE environment. Interaction confidence was ranked according to the NWD scores ( Sowa et al., 2009 ). The results obtained from the CompPASS analysis were filtered to determine proteins with a NWD score > 1 or high confidence interactors (HCIPs). HCIPs were then filtered for mitochondrial proteins based on the proteins present in Mitocarta ( Pagliarini et al., 2008 ). For the 293T data set, HCIPs with 2 or more ASPMs were included. DOI: http://dx.doi.org/10.7554/eLife.06265.005

    Techniques Used: Mass Spectrometry

    2) Product Images from "Pepitome: evaluating improved spectral library search for identification complementarity and quality assessment"

    Article Title: Pepitome: evaluating improved spectral library search for identification complementarity and quality assessment

    Journal: Journal of Proteome Research

    doi: 10.1021/pr200874e

    Quality Assessment (QA) Method for Shotgun Proteomics Data Sets We performed 902 LC-MS/MS analyses of BSA standards on a LTQ-XL instrument spanning 18 months. Experts reviewed the raw files and assigned a quality label (low or high) to each file. Pepitome identified peptides from the samples and IDPicker filtered the identifications at 2% FDR (a) Peptide and spectral identification rates from quality assessed raw files. (b) We developed an artificial neural network (ANN) for recapitulating the expert QA of a raw file from a collection of quality metrics. This figure shows the training and testing receiver operating curves when the ANN is employing different categorical collections of quality metrics as inputs.
    Figure Legend Snippet: Quality Assessment (QA) Method for Shotgun Proteomics Data Sets We performed 902 LC-MS/MS analyses of BSA standards on a LTQ-XL instrument spanning 18 months. Experts reviewed the raw files and assigned a quality label (low or high) to each file. Pepitome identified peptides from the samples and IDPicker filtered the identifications at 2% FDR (a) Peptide and spectral identification rates from quality assessed raw files. (b) We developed an artificial neural network (ANN) for recapitulating the expert QA of a raw file from a collection of quality metrics. This figure shows the training and testing receiver operating curves when the ANN is employing different categorical collections of quality metrics as inputs.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Related Articles

    Mass Spectrometry:

    Article Title: Elucidation and Chemical Modulation of Sulfolipid-1 Biosynthesis in Mycobacterium tuberculosis *
    Article Snippet: .. For the mass range m /z 300–1000, the capillary voltage was set to 4.5 kV, the capillary exit voltage was set to −300 V, the skimmer 1 voltage was set to −20 V, and the skimmer 2 voltage was set to −7 V. For the mass range m /z 1000–3000, the skimmer 2 voltage was lowered to approximately −1 to −3 V. Additional MSn spectra were obtained on an LTQ mass spectrometer equipped with an electrospray ionization source (Thermo Finnigan) operating in the negative ion mode. .. Ions were introduced into the ion source via direct injection at a rate of 5–10 μl/min.

    Article Title: QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology
    Article Snippet: .. Samples were ran in technical duplicate on either an Thermo LTQ mass spectrometer or an LTQ-Orbitrap Elite, and spectra search with Sequest prior to target-decoy peptide filtering, and linear discriminant analysis ( ). ..

    Article Title: Conformation-selective inhibitors reveal differences in the activation and phosphate-binding loops of the tyrosine kinases Abl and Src
    Article Snippet: .. Samples were analyzed on a Thermo Finnigan LTQ mass spectrometer. ..

    Article Title: The gut microbiota modulates host amino acid and glutathione metabolism in mice
    Article Snippet: .. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) portion of the platform was based on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo-Finnigan LTQ mass spectrometer operated at nominal mass resolution, which consisted of an electrospray ionization (ESI) source and linear ion-trap (LIT) mass analyzer. .. The sample extract was dried and then reconstituted in acidic or basic LC-compatible solvents, each of which contained 12 or more injection standards at fixed concentrations.

    Article Title: Pepitome: evaluating improved spectral library search for identification complementarity and quality assessment
    Article Snippet: .. The 1X BSA peptide mixtures were analyzed on a Thermo Fisher LTQ mass spectrometer (manufacturer serial no. LTQ20585; alias: Amigo-2) equipped with an Eksigent nanoLC and autosampler (Dublin, CA). .. Peptides were resolved on a 100μm × 11cm fused silica capillary column (Polymicro Technologies, Phoenix, AZ) packed with 5μm, 300Å Jupiter C18 resin (Phenomenex, Torrance, CA) and an inline 100mm × 4cm solid phase extraction column packed with the same C18 resin .

    Article Title: Rapamycin regulates biochemical metabolites
    Article Snippet: .. The LC/MS portion of the platform was based on a Waters ACQUITY UPLC and a Thermo-Finnigan LTQ mass spectrometer, which consisted of an electrospray ionization source and linear ion-trap mass analyzer. .. The sample extract was split into two aliquots, dried, and then reconstituted in acidic or basic LC-compatible solvents, each of which contained 11 or more injection standards at fixed concentrations.

    Article Title: Covalent Peroxisome Proliferator-activated Receptor ? Adduction by Nitro-fatty Acids
    Article Snippet: .. For quantification, the LTQ mass spectrometer was set to continuously isolate and fragment the specific m/z values per period of the different peptides of interest (up to 15 ions per period). ..

    Article Title: Metabolic adaptation of short-living growth hormone transgenic mice to methionine restriction and supplementation
    Article Snippet: .. Liquid chromatography/mass spectrometry (LC/MS) was performed using a Waters Acquity UPLC system and a Thermo-Finnigan LTQ mass spectrometer, including an electrospray ionization source and linear ion-trap (LIT) mass analyzer. .. Aliquots of vacuum-dried samples were reconstituted, one each in acidic or basic LC-compatible solvents containing 8 or more injection standards at fixed concentrations (to both ensure injection and chromatographic consistency).

    Chromatography:

    Article Title: The gut microbiota modulates host amino acid and glutathione metabolism in mice
    Article Snippet: .. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) portion of the platform was based on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo-Finnigan LTQ mass spectrometer operated at nominal mass resolution, which consisted of an electrospray ionization (ESI) source and linear ion-trap (LIT) mass analyzer. .. The sample extract was dried and then reconstituted in acidic or basic LC-compatible solvents, each of which contained 12 or more injection standards at fixed concentrations.

    Article Title: Metabolic adaptation of short-living growth hormone transgenic mice to methionine restriction and supplementation
    Article Snippet: .. Liquid chromatography/mass spectrometry (LC/MS) was performed using a Waters Acquity UPLC system and a Thermo-Finnigan LTQ mass spectrometer, including an electrospray ionization source and linear ion-trap (LIT) mass analyzer. .. Aliquots of vacuum-dried samples were reconstituted, one each in acidic or basic LC-compatible solvents containing 8 or more injection standards at fixed concentrations (to both ensure injection and chromatographic consistency).

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Rapamycin regulates biochemical metabolites
    Article Snippet: .. The LC/MS portion of the platform was based on a Waters ACQUITY UPLC and a Thermo-Finnigan LTQ mass spectrometer, which consisted of an electrospray ionization source and linear ion-trap mass analyzer. .. The sample extract was split into two aliquots, dried, and then reconstituted in acidic or basic LC-compatible solvents, each of which contained 11 or more injection standards at fixed concentrations.

    Article Title: Metabolic adaptation of short-living growth hormone transgenic mice to methionine restriction and supplementation
    Article Snippet: .. Liquid chromatography/mass spectrometry (LC/MS) was performed using a Waters Acquity UPLC system and a Thermo-Finnigan LTQ mass spectrometer, including an electrospray ionization source and linear ion-trap (LIT) mass analyzer. .. Aliquots of vacuum-dried samples were reconstituted, one each in acidic or basic LC-compatible solvents containing 8 or more injection standards at fixed concentrations (to both ensure injection and chromatographic consistency).

    Liquid Chromatography:

    Article Title: The gut microbiota modulates host amino acid and glutathione metabolism in mice
    Article Snippet: .. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) portion of the platform was based on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo-Finnigan LTQ mass spectrometer operated at nominal mass resolution, which consisted of an electrospray ionization (ESI) source and linear ion-trap (LIT) mass analyzer. .. The sample extract was dried and then reconstituted in acidic or basic LC-compatible solvents, each of which contained 12 or more injection standards at fixed concentrations.

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    Thermo Fisher ltq orbitrap mass spectrometer
    Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an <t>LTQ-Orbitrap</t> mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.
    Ltq Orbitrap Mass Spectrometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ltq orbitrap mass spectrometer/product/Thermo Fisher
    Average 92 stars, based on 436 article reviews
    Price from $9.99 to $1999.99
    ltq orbitrap mass spectrometer - by Bioz Stars, 2020-05
    92/100 stars
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    93
    Thermo Fisher ltq orbitrap velos mass spectrometer
    A schematic workflow illustrating the steps involved in the differential analysis of RA and OA synovial fluid proteome. Proteins from RA and OA synovial fluid were extracted and depleted to remove the 14 most abundant proteins using multiple affinity removal system, Human-14. The depleted protein from RA and OA were then digested with trypsin and labeled with iTRAQ reagents, 117 and 116 respectively. The labeled samples were pooled and subjected to fractionation using strong cation exchange chromatography. The fractions were then analyzed on a <t>LTQ-Orbitrap</t> <t>Velos</t> mass spectrometer. The MS/MS data obtained was searched against Human RefSeq 50 database using Sequest and Mascot search algorithms. Validation of the iTRAQ quantitation data was carried out using multiple reaction monitoring and Western blot.
    Ltq Orbitrap Velos Mass Spectrometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ltq orbitrap velos mass spectrometer/product/Thermo Fisher
    Average 93 stars, based on 81 article reviews
    Price from $9.99 to $1999.99
    ltq orbitrap velos mass spectrometer - by Bioz Stars, 2020-05
    93/100 stars
      Buy from Supplier

    Image Search Results


    Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.

    Journal: Open Biology

    Article Title: PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

    doi: 10.1098/rsob.120080

    Figure Lengend Snippet: Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.

    Article Snippet: Isolated phosphopeptides were analysed by LC-MS-MS on a proxeon Easy-nLC nano liquid chromatography system coupled to a Thermo LTQ-orbitrap mass spectrometer.

    Techniques: Mass Spectrometry, Activation Assay, Expressing, Transfection, Lysis, Immunoprecipitation, SDS Page, Staining, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

    Schematic diagram of proteomic analysis of the mouse organ of Corti (OC) sample. ( 1 ) The OCs were reconstituted in 100 μL lysis buffer (100 mM ammonium bicarbonate, pH 8.4); ( 2 ) The lysates were reduced by 5 mM DL-Dithiothreitol (DTT) and digested by trypsin overnight; ( 3 ) The digests were desalted and dried in a vacuum centrifuge immediately after digestion; ( 4 ) Dried peptides were subjected to the in-house assembled reverse phase metal-free multiple-column nanoLC system coupled with ( 5 ) LTQ-Orbitrap XL mass spectrometer for MS analysis.

    Journal: International Journal of Molecular Sciences

    Article Title: Proteomic Analysis of the Organ of Corti Using Nanoscale Liquid Chromatography Coupled with Tandem Mass Spectrometry

    doi: 10.3390/ijms13078171

    Figure Lengend Snippet: Schematic diagram of proteomic analysis of the mouse organ of Corti (OC) sample. ( 1 ) The OCs were reconstituted in 100 μL lysis buffer (100 mM ammonium bicarbonate, pH 8.4); ( 2 ) The lysates were reduced by 5 mM DL-Dithiothreitol (DTT) and digested by trypsin overnight; ( 3 ) The digests were desalted and dried in a vacuum centrifuge immediately after digestion; ( 4 ) Dried peptides were subjected to the in-house assembled reverse phase metal-free multiple-column nanoLC system coupled with ( 5 ) LTQ-Orbitrap XL mass spectrometer for MS analysis.

    Article Snippet: The nanoLC system was interfaced with a LTQ-Orbitrap XL mass spectrometer equipped with a nanoelectrospray ion source (Thermo Scientific, Waltham, MA, USA).

    Techniques: Lysis, Mass Spectrometry

    Quantification of calcium-dependent regulation of phosphorylation sites of Toxoplasma invasion motor complex components. A ) Work flow to identify individual phosphorylation sites and quantitatively assess their responsiveness to calcium signals using a SILAC-based proteomics approach. A 1∶1 mixture of Triton X-100 lysates from “Heavy” (H; Arg4/Lys8)-labeled ethanol-stimulated tachyzoites or

    Journal: PLoS Pathogens

    Article Title: Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

    doi: 10.1371/journal.ppat.1002222

    Figure Lengend Snippet: Quantification of calcium-dependent regulation of phosphorylation sites of Toxoplasma invasion motor complex components. A ) Work flow to identify individual phosphorylation sites and quantitatively assess their responsiveness to calcium signals using a SILAC-based proteomics approach. A 1∶1 mixture of Triton X-100 lysates from “Heavy” (H; Arg4/Lys8)-labeled ethanol-stimulated tachyzoites or "Light" (L; Arg0/Lys0)-labeled non-stimulated parasites was generated, and a TiO 2 -enriched phosphopeptide sample of H/L-labeled Toxoplasma invasion motor complexes was prepared and analysed by LC-MS/MS on an LTQ-Orbitrap instrument. Mascot and MaxQuant search engines facilitated subsequent manual identification, phosphosite localization and quantification of proteins or peptides as detailed in materials and mathods. B ) Sypro Ruby-stained SDS-PAGE separation of the relative amounts of light (lane 1) or heavy (lane 2) Triton X-100 whole protein extracts are shown. Intact tachyzoite invasion motor complexes comprising the five major components MyoA, GAP50, GAP45 and MLC1 were precipitated from a 1∶1 H/L mixture by GAP45-specific immuno-affinity chromatography (lane 3).

    Article Snippet: The nano HPLC was coupled on-line to an LTQ-Orbitrap mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific) for automated MS/MS.

    Techniques: Flow Cytometry, Labeling, Generated, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Staining, SDS Page, Affinity Chromatography

    A schematic workflow illustrating the steps involved in the differential analysis of RA and OA synovial fluid proteome. Proteins from RA and OA synovial fluid were extracted and depleted to remove the 14 most abundant proteins using multiple affinity removal system, Human-14. The depleted protein from RA and OA were then digested with trypsin and labeled with iTRAQ reagents, 117 and 116 respectively. The labeled samples were pooled and subjected to fractionation using strong cation exchange chromatography. The fractions were then analyzed on a LTQ-Orbitrap Velos mass spectrometer. The MS/MS data obtained was searched against Human RefSeq 50 database using Sequest and Mascot search algorithms. Validation of the iTRAQ quantitation data was carried out using multiple reaction monitoring and Western blot.

    Journal: Clinical proteomics

    Article Title: Differential proteomic analysis of synovial fluid from rheumatoid arthritis and osteoarthritis patients

    doi: 10.1186/1559-0275-11-1

    Figure Lengend Snippet: A schematic workflow illustrating the steps involved in the differential analysis of RA and OA synovial fluid proteome. Proteins from RA and OA synovial fluid were extracted and depleted to remove the 14 most abundant proteins using multiple affinity removal system, Human-14. The depleted protein from RA and OA were then digested with trypsin and labeled with iTRAQ reagents, 117 and 116 respectively. The labeled samples were pooled and subjected to fractionation using strong cation exchange chromatography. The fractions were then analyzed on a LTQ-Orbitrap Velos mass spectrometer. The MS/MS data obtained was searched against Human RefSeq 50 database using Sequest and Mascot search algorithms. Validation of the iTRAQ quantitation data was carried out using multiple reaction monitoring and Western blot.

    Article Snippet: LC-MS/MS analysis Tandem mass spectrometric analysis of the iTRAQ labeled peptides were carried out using LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, Bremen, Germany) interfaced with Easy nanoLC II (previously Proxeon, Thermo Scientific, Bremen, Germany).

    Techniques: Labeling, Fractionation, Chromatography, Mass Spectrometry, Quantitation Assay, Western Blot