nanolc system  (Thermo Fisher)


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    Structured Review

    Thermo Fisher nanolc system
    ( A ) Schematic representation of the workflow used to identify SLeA-expressing glycoproteins with affinity for E-selectin. Briefly, IPs with a monoclonal targeting the SLeA antigen were used to isolate glycoproteins from plasma membrane glycoproteins enriched extracts. In parallel, samples were pulled down with E-selectin. Parallel IPs with isotype controls and lectin pulldowns in the absence of Ca 2+ ions (required for binding) were also performed as negative controls. The proteins were then resolved by SDS-PAGE, the bands were excised, proteins reduced, alkylated and digested with trypsin prior to analysis by <t>nanoLC-ESI-MS/MS.</t> ( B ) SLeA and SLeX expressions in glycoproteins obtained by IP for SLeA and corresponding isotype controls for N87 (SLeA+/SLeX+) and AGS (SLeA-/SLeX-; negative control) as well as glycoproteins with affinity for E-selectin in the presence and absence of Ca2+ (negative control). Collectively, the blots support the affinity of the IP strategy for SLeA-expressing glycoproteins with no contaminations from SLeX glycoproteins. On the other hand, E-selectin isolated both SLeA and SLeX expressing glycoproteins. ( C ) Venn diagrams highlighting the distribution SLeA-expressing glycoproteins and glycoproteins showing affinity for human E-selectin for cell line N87 ( A ), OCUM-1 ( B ), and KATO-III ( C ). Glycoproteins with confirmed O-SLeA expressing glycosites and showing affinity for E-selectin among the three cell models ( D ). The Venn diagrams in panels A–C highlight the number of glycoproteins isolated by IP for SLeA and E-selectin for each cell line, evidencing the percentage of glycoproteins commonly identified for both IPs and showing clear SLeA modifications in O-glycans. Panel C highlights the 22 glycoproteins presenting O-SLeA glycosylation and affinity for E-selectin in all cell lines.
    Nanolc System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Nucleolin-Sle A Glycoforms as E-Selectin Ligands and Potentially Targetable Biomarkers at the Cell Surface of Gastric Cancer Cells"

    Article Title: Nucleolin-Sle A Glycoforms as E-Selectin Ligands and Potentially Targetable Biomarkers at the Cell Surface of Gastric Cancer Cells

    Journal: Cancers

    doi: 10.3390/cancers12040861

    ( A ) Schematic representation of the workflow used to identify SLeA-expressing glycoproteins with affinity for E-selectin. Briefly, IPs with a monoclonal targeting the SLeA antigen were used to isolate glycoproteins from plasma membrane glycoproteins enriched extracts. In parallel, samples were pulled down with E-selectin. Parallel IPs with isotype controls and lectin pulldowns in the absence of Ca 2+ ions (required for binding) were also performed as negative controls. The proteins were then resolved by SDS-PAGE, the bands were excised, proteins reduced, alkylated and digested with trypsin prior to analysis by nanoLC-ESI-MS/MS. ( B ) SLeA and SLeX expressions in glycoproteins obtained by IP for SLeA and corresponding isotype controls for N87 (SLeA+/SLeX+) and AGS (SLeA-/SLeX-; negative control) as well as glycoproteins with affinity for E-selectin in the presence and absence of Ca2+ (negative control). Collectively, the blots support the affinity of the IP strategy for SLeA-expressing glycoproteins with no contaminations from SLeX glycoproteins. On the other hand, E-selectin isolated both SLeA and SLeX expressing glycoproteins. ( C ) Venn diagrams highlighting the distribution SLeA-expressing glycoproteins and glycoproteins showing affinity for human E-selectin for cell line N87 ( A ), OCUM-1 ( B ), and KATO-III ( C ). Glycoproteins with confirmed O-SLeA expressing glycosites and showing affinity for E-selectin among the three cell models ( D ). The Venn diagrams in panels A–C highlight the number of glycoproteins isolated by IP for SLeA and E-selectin for each cell line, evidencing the percentage of glycoproteins commonly identified for both IPs and showing clear SLeA modifications in O-glycans. Panel C highlights the 22 glycoproteins presenting O-SLeA glycosylation and affinity for E-selectin in all cell lines.
    Figure Legend Snippet: ( A ) Schematic representation of the workflow used to identify SLeA-expressing glycoproteins with affinity for E-selectin. Briefly, IPs with a monoclonal targeting the SLeA antigen were used to isolate glycoproteins from plasma membrane glycoproteins enriched extracts. In parallel, samples were pulled down with E-selectin. Parallel IPs with isotype controls and lectin pulldowns in the absence of Ca 2+ ions (required for binding) were also performed as negative controls. The proteins were then resolved by SDS-PAGE, the bands were excised, proteins reduced, alkylated and digested with trypsin prior to analysis by nanoLC-ESI-MS/MS. ( B ) SLeA and SLeX expressions in glycoproteins obtained by IP for SLeA and corresponding isotype controls for N87 (SLeA+/SLeX+) and AGS (SLeA-/SLeX-; negative control) as well as glycoproteins with affinity for E-selectin in the presence and absence of Ca2+ (negative control). Collectively, the blots support the affinity of the IP strategy for SLeA-expressing glycoproteins with no contaminations from SLeX glycoproteins. On the other hand, E-selectin isolated both SLeA and SLeX expressing glycoproteins. ( C ) Venn diagrams highlighting the distribution SLeA-expressing glycoproteins and glycoproteins showing affinity for human E-selectin for cell line N87 ( A ), OCUM-1 ( B ), and KATO-III ( C ). Glycoproteins with confirmed O-SLeA expressing glycosites and showing affinity for E-selectin among the three cell models ( D ). The Venn diagrams in panels A–C highlight the number of glycoproteins isolated by IP for SLeA and E-selectin for each cell line, evidencing the percentage of glycoproteins commonly identified for both IPs and showing clear SLeA modifications in O-glycans. Panel C highlights the 22 glycoproteins presenting O-SLeA glycosylation and affinity for E-selectin in all cell lines.

    Techniques Used: Expressing, Binding Assay, SDS Page, Tandem Mass Spectroscopy, Negative Control, Isolation

    2) Product Images from "Proteomic Analysis of the Organ of Corti Using Nanoscale Liquid Chromatography Coupled with Tandem Mass Spectrometry"

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

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms13078171

    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.
    Figure Legend 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.

    Techniques Used: Lysis, Mass Spectrometry

    3) Product Images from "Targeted O‐glycoproteomics explored increased sialylation and identified MUC16 as a poor prognosis biomarker in advanced‐stage bladder tumours"

    Article Title: Targeted O‐glycoproteomics explored increased sialylation and identified MUC16 as a poor prognosis biomarker in advanced‐stage bladder tumours

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12035

    (A) Exemplificative annotated nanoLC‐ESI‐LTQ‐Orbitrap‐CID‐MS/MS spectra for a MUC16 glycopeptide substituted with a HexNAc residue evidencing the specific glycosite; (B) colocalization of MUC16 and STn in bladder tumours by immunohistochemistry; (C) expression of MUC16 STn glycoforms in bladder tumours based on PLA analysis. This work identified for the first time MUC16 in bladder tumours and its association with abnormal glycoforms such as the STn antigen. The mass spectrum shows a MUC16 glycopeptide substituted with a HexNAc residue, strongly suggesting the presence of STn. The colocalization of MUC16 and STn (B) in bladder tumours also reinforces this hypothesis. Finally, the red dots on the PLA image (C) in areas of colocalization result from the simultaneous detection of both antigens, reinforcing this evidence.
    Figure Legend Snippet: (A) Exemplificative annotated nanoLC‐ESI‐LTQ‐Orbitrap‐CID‐MS/MS spectra for a MUC16 glycopeptide substituted with a HexNAc residue evidencing the specific glycosite; (B) colocalization of MUC16 and STn in bladder tumours by immunohistochemistry; (C) expression of MUC16 STn glycoforms in bladder tumours based on PLA analysis. This work identified for the first time MUC16 in bladder tumours and its association with abnormal glycoforms such as the STn antigen. The mass spectrum shows a MUC16 glycopeptide substituted with a HexNAc residue, strongly suggesting the presence of STn. The colocalization of MUC16 and STn (B) in bladder tumours also reinforces this hypothesis. Finally, the red dots on the PLA image (C) in areas of colocalization result from the simultaneous detection of both antigens, reinforcing this evidence.

    Techniques Used: Mass Spectrometry, Immunohistochemistry, Expressing, Proximity Ligation Assay

    Related Articles

    Mass Spectrometry:

    Article Title: FUS-dependent phase separation initiates double-strand break repair
    Article Snippet: .. Mass spectrometry analysis was performed by nano-liquid chromatography–tandem MS (nLC–ESI-MS/MS). .. Peptides separation was achieved on a linear gradient from 95% solvent A (2% ACN, 0.1% formic acid) to 40% solvent B (80% acetonitrile, 0.1% formic acid) over 1:30 h and from 40% to 100% solvent B in 2 min at a constant flow rate of 250 nl/min on UHPLC Easy-nLC 1000 (Thermo Scientific) using high pressure bomb loader.

    Chromatography:

    Article Title: FUS-dependent phase separation initiates double-strand break repair
    Article Snippet: .. Mass spectrometry analysis was performed by nano-liquid chromatography–tandem MS (nLC–ESI-MS/MS). .. Peptides separation was achieved on a linear gradient from 95% solvent A (2% ACN, 0.1% formic acid) to 40% solvent B (80% acetonitrile, 0.1% formic acid) over 1:30 h and from 40% to 100% solvent B in 2 min at a constant flow rate of 250 nl/min on UHPLC Easy-nLC 1000 (Thermo Scientific) using high pressure bomb loader.

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    Thermo Fisher esi nanolc ms ms analysis
    Workflow of the experimental strategy used in this study. Plasma samples from six experimental groups were trypsin digested, labeled with isobaric tags, pooled and then purified and fractionated using SCX method. Quantitative proteomic analyses were simultaneously performed using <t>ESI-nanoLC-MS/MS</t> and MALDI-nanoLC-MS/MS and then obtained data were analyzed with three types of software: MaxQuant, ProteinScape and Proteome Discoverer. Only proteins identified by all software were found to have a differential accumulation level.
    Esi Nanolc Ms Ms Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher nf1 nanoluc pcdna expression plasmid
    AMG-510 prevents KRAS-G12C from interacting with <t>NF1</t> and CRAF. ( A ) SW48 isogenic cell lines, SW837 and SW1463 were treated with increasing doses of AMG-510 for 48 hours and cell viability was measured with the MTT assay. Data points represent the mean of eight biological replicates. Results are representative of three separate experiments. ( B ) HEK293T cells were co-transfected with KRAS-GFP and <t>NF1-NanoLuc</t> with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( C ) HEK293T cells were co-transfected with KRAS-GFP and CRAF-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( D ). HEK293T cells were transfected with either KRAS-G12C-GFP and NF1, NF1 alone, or mock transfection. Cells were then treated with either vehicle or 500 nM AMG-510 for 24 hours. Results are representative of an individual experiment from three separate experiments. ( E ) Mean KRAS G12C pull-down for the three independent NF1-coIP experiments. Statistical analysis was performed with unpaired t-test and P-values are indicated. Error bars in all panels represent standard deviation.
    Nf1 Nanoluc Pcdna Expression Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher nanolc qtof ms all
    <t>NanoLC-qTOF-MS</t> glycopeptide profiling of IgG and IgA. (A) Extracted ion chromatograms (EICs) for one major glycopeptide per cluster. Protein names and the first three letters of the amino acid sequence of the respective tryptic peptide (in parentheses) are given. Separation was based on the peptide backbones, clustering the analytes with the same peptide sequence, but varying glycan portions. The m / z window for the EICs was set as follows for the different clusters: ± 0.05 Th for SES, ± 0.002 Th for IgG4, ± 0.01 Th for ENI, and ±0.02 Th for all other clusters. The blue and red boxes indicate the time windows used for sum spectra generation in (B) and (C). (B) The 25 most abundant glycopeptide peaks from the IgA1 HYT O -glycopeptide cluster, their measured m / z and suggested monosaccharide compositions. (C) The 20 most abundant glycopeptide peaks from the IgA1/2 LSL N -glycopeptide cluster, their measured m / z and proposed N -glycan structures. 25 , 37 Note: linkages were not determined. Asterisks mark signals not derived from glycopeptides.
    Nanolc Qtof Ms All, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nanolc ms
    Accumulation of unique peptide spectral matches with replicate experiments. The number of unique peptides identified is shown with respect to the number of combined <t>nanoLC-FAIMS-MS</t> experiments at different compensation voltages (squares) or combined replicate
    Nanolc Ms, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Workflow of the experimental strategy used in this study. Plasma samples from six experimental groups were trypsin digested, labeled with isobaric tags, pooled and then purified and fractionated using SCX method. Quantitative proteomic analyses were simultaneously performed using ESI-nanoLC-MS/MS and MALDI-nanoLC-MS/MS and then obtained data were analyzed with three types of software: MaxQuant, ProteinScape and Proteome Discoverer. Only proteins identified by all software were found to have a differential accumulation level.

    Journal: Scientific Reports

    Article Title: iTRAQ-based proteomic analysis of plasma reveals abnormalities in lipid metabolism proteins in chronic kidney disease-related atherosclerosis

    doi: 10.1038/srep32511

    Figure Lengend Snippet: Workflow of the experimental strategy used in this study. Plasma samples from six experimental groups were trypsin digested, labeled with isobaric tags, pooled and then purified and fractionated using SCX method. Quantitative proteomic analyses were simultaneously performed using ESI-nanoLC-MS/MS and MALDI-nanoLC-MS/MS and then obtained data were analyzed with three types of software: MaxQuant, ProteinScape and Proteome Discoverer. Only proteins identified by all software were found to have a differential accumulation level.

    Article Snippet: ESI-NanoLC-MS/MS analysis For each iTRAQ experiment, 5 μ l of respectively KCl fraction was injected on a RP C18 precolumn (Thermo Fisher Scientific, USA) connected to a 75-μ m i.d.

    Techniques: Labeling, Purification, Mass Spectrometry, Software

    AMG-510 prevents KRAS-G12C from interacting with NF1 and CRAF. ( A ) SW48 isogenic cell lines, SW837 and SW1463 were treated with increasing doses of AMG-510 for 48 hours and cell viability was measured with the MTT assay. Data points represent the mean of eight biological replicates. Results are representative of three separate experiments. ( B ) HEK293T cells were co-transfected with KRAS-GFP and NF1-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( C ) HEK293T cells were co-transfected with KRAS-GFP and CRAF-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( D ). HEK293T cells were transfected with either KRAS-G12C-GFP and NF1, NF1 alone, or mock transfection. Cells were then treated with either vehicle or 500 nM AMG-510 for 24 hours. Results are representative of an individual experiment from three separate experiments. ( E ) Mean KRAS G12C pull-down for the three independent NF1-coIP experiments. Statistical analysis was performed with unpaired t-test and P-values are indicated. Error bars in all panels represent standard deviation.

    Journal: bioRxiv

    Article Title: Inhibition of both mutant and wild-type RAS-GTP in KRAS G12C colorectal cancer through cotreatment with G12C and EGFR inhibitors

    doi: 10.1101/845263

    Figure Lengend Snippet: AMG-510 prevents KRAS-G12C from interacting with NF1 and CRAF. ( A ) SW48 isogenic cell lines, SW837 and SW1463 were treated with increasing doses of AMG-510 for 48 hours and cell viability was measured with the MTT assay. Data points represent the mean of eight biological replicates. Results are representative of three separate experiments. ( B ) HEK293T cells were co-transfected with KRAS-GFP and NF1-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( C ) HEK293T cells were co-transfected with KRAS-GFP and CRAF-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( D ). HEK293T cells were transfected with either KRAS-G12C-GFP and NF1, NF1 alone, or mock transfection. Cells were then treated with either vehicle or 500 nM AMG-510 for 24 hours. Results are representative of an individual experiment from three separate experiments. ( E ) Mean KRAS G12C pull-down for the three independent NF1-coIP experiments. Statistical analysis was performed with unpaired t-test and P-values are indicated. Error bars in all panels represent standard deviation.

    Article Snippet: Twenty-four hours after seeding, cells were co-transfected with either a constant concentration of 0.1 μg of NF1-NanoLuc pcDNA expression plasmid or CRAF RBD-NanoLuc pcDNA expression plasmid with increasing concentrations of GFP-tagged KRAS (WT or Mutant) with 0.25 μl of Lipofectamine 2000 per well following the manufacturer’s protocol (Thermo Fisher Scientific).

    Techniques: MTT Assay, Transfection, Bioluminescence Resonance Energy Transfer, Co-Immunoprecipitation Assay, Standard Deviation

    KRAS-G12C binds less well to NF1 and to CRAF. ( A ) HEK293T cells were transfected with increasing amount of KRAS-GFP expression plasmid and a constant concentration of NF1-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with eight biological replicates, and were repeated three times. ( B ) Average BRET ratio from the three experiments at a RAS:NF1 ratio of 4:1. ( C ) HEK293T cells were transfected with either WT-KRAS-GFP, G12V-KRAS-GFP, G12C-KRAS GFP or NF1-FLAG expression plasmids. The NF1 co-IP mixing assay was performed and IP product and input lysate were probed by western blot for NF1, KRAS, and GFP. Results are representative of three independent experiments. ( D ) Average intensity of NF1 co-IP from the three independent experiments. ( E ) Isoelectric focused whole cell lysates from SW48 G12C, SW48 G12V and SW48 WT isogenic cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( F ) Average proportions of KRAS, NRAS, and HRAS bound to RBD and in whole cell lysates from three separate IEF experiments. ( G ) Isoelectric focused whole cell lysates from heterozygous mutant KRAS G12C SW837 and homozygous mutant KRAS G12C SW1463 cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( H ) Average proportions of KRAS, NRAS, and HRAS bound to and in whole cell lysates from three separate IEF experiments. ( I ) HEK293T cells were transfected with increasing concentrations of KRAS-GFP expression plasmids and a constant concentration of CRAF-RBD-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with three experimental replicates, each containing eight biological replicates. ( J ) Average BRET ratio from the three experiments at a RAS:RBD ratio of 2:1. ( K ) SW48 Isogenic cells were harvested and prepared for RAF-RBD pulldown assay, one set was not exposed to γ-S-GTP and the other was incubated in γ-S-GTP for 20 minutes. IP-product and input were probed by western blot for KRAS and GAPDH. Results are representative of three independent experiments. ( L ) Average quantity of KRAS WT, KRAS G12C, and KRAS G12C pulled down by RAF-RBD from three independent experiments. All experiments were performed three times. Treatment groups were analyzed by one-way ANOVA followed by post-hoc Tukey’s test for multiple comparisons and P-values are indicated. Error bars in all panels represent standard deviation.

    Journal: bioRxiv

    Article Title: Inhibition of both mutant and wild-type RAS-GTP in KRAS G12C colorectal cancer through cotreatment with G12C and EGFR inhibitors

    doi: 10.1101/845263

    Figure Lengend Snippet: KRAS-G12C binds less well to NF1 and to CRAF. ( A ) HEK293T cells were transfected with increasing amount of KRAS-GFP expression plasmid and a constant concentration of NF1-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with eight biological replicates, and were repeated three times. ( B ) Average BRET ratio from the three experiments at a RAS:NF1 ratio of 4:1. ( C ) HEK293T cells were transfected with either WT-KRAS-GFP, G12V-KRAS-GFP, G12C-KRAS GFP or NF1-FLAG expression plasmids. The NF1 co-IP mixing assay was performed and IP product and input lysate were probed by western blot for NF1, KRAS, and GFP. Results are representative of three independent experiments. ( D ) Average intensity of NF1 co-IP from the three independent experiments. ( E ) Isoelectric focused whole cell lysates from SW48 G12C, SW48 G12V and SW48 WT isogenic cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( F ) Average proportions of KRAS, NRAS, and HRAS bound to RBD and in whole cell lysates from three separate IEF experiments. ( G ) Isoelectric focused whole cell lysates from heterozygous mutant KRAS G12C SW837 and homozygous mutant KRAS G12C SW1463 cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( H ) Average proportions of KRAS, NRAS, and HRAS bound to and in whole cell lysates from three separate IEF experiments. ( I ) HEK293T cells were transfected with increasing concentrations of KRAS-GFP expression plasmids and a constant concentration of CRAF-RBD-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with three experimental replicates, each containing eight biological replicates. ( J ) Average BRET ratio from the three experiments at a RAS:RBD ratio of 2:1. ( K ) SW48 Isogenic cells were harvested and prepared for RAF-RBD pulldown assay, one set was not exposed to γ-S-GTP and the other was incubated in γ-S-GTP for 20 minutes. IP-product and input were probed by western blot for KRAS and GAPDH. Results are representative of three independent experiments. ( L ) Average quantity of KRAS WT, KRAS G12C, and KRAS G12C pulled down by RAF-RBD from three independent experiments. All experiments were performed three times. Treatment groups were analyzed by one-way ANOVA followed by post-hoc Tukey’s test for multiple comparisons and P-values are indicated. Error bars in all panels represent standard deviation.

    Article Snippet: Twenty-four hours after seeding, cells were co-transfected with either a constant concentration of 0.1 μg of NF1-NanoLuc pcDNA expression plasmid or CRAF RBD-NanoLuc pcDNA expression plasmid with increasing concentrations of GFP-tagged KRAS (WT or Mutant) with 0.25 μl of Lipofectamine 2000 per well following the manufacturer’s protocol (Thermo Fisher Scientific).

    Techniques: Transfection, Expressing, Plasmid Preparation, Concentration Assay, Bioluminescence Resonance Energy Transfer, Co-Immunoprecipitation Assay, Western Blot, Electrofocusing, Mutagenesis, Incubation, Standard Deviation

    NanoLC-qTOF-MS glycopeptide profiling of IgG and IgA. (A) Extracted ion chromatograms (EICs) for one major glycopeptide per cluster. Protein names and the first three letters of the amino acid sequence of the respective tryptic peptide (in parentheses) are given. Separation was based on the peptide backbones, clustering the analytes with the same peptide sequence, but varying glycan portions. The m / z window for the EICs was set as follows for the different clusters: ± 0.05 Th for SES, ± 0.002 Th for IgG4, ± 0.01 Th for ENI, and ±0.02 Th for all other clusters. The blue and red boxes indicate the time windows used for sum spectra generation in (B) and (C). (B) The 25 most abundant glycopeptide peaks from the IgA1 HYT O -glycopeptide cluster, their measured m / z and suggested monosaccharide compositions. (C) The 20 most abundant glycopeptide peaks from the IgA1/2 LSL N -glycopeptide cluster, their measured m / z and proposed N -glycan structures. 25 , 37 Note: linkages were not determined. Asterisks mark signals not derived from glycopeptides.

    Journal: Analytical Chemistry

    Article Title: Simultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications

    doi: 10.1021/acs.analchem.9b05722

    Figure Lengend Snippet: NanoLC-qTOF-MS glycopeptide profiling of IgG and IgA. (A) Extracted ion chromatograms (EICs) for one major glycopeptide per cluster. Protein names and the first three letters of the amino acid sequence of the respective tryptic peptide (in parentheses) are given. Separation was based on the peptide backbones, clustering the analytes with the same peptide sequence, but varying glycan portions. The m / z window for the EICs was set as follows for the different clusters: ± 0.05 Th for SES, ± 0.002 Th for IgG4, ± 0.01 Th for ENI, and ±0.02 Th for all other clusters. The blue and red boxes indicate the time windows used for sum spectra generation in (B) and (C). (B) The 25 most abundant glycopeptide peaks from the IgA1 HYT O -glycopeptide cluster, their measured m / z and suggested monosaccharide compositions. (C) The 20 most abundant glycopeptide peaks from the IgA1/2 LSL N -glycopeptide cluster, their measured m / z and proposed N -glycan structures. 25 , 37 Note: linkages were not determined. Asterisks mark signals not derived from glycopeptides.

    Article Snippet: Glycosylation Profiling by NanoLC-qTOF-MS All samples were thawed, mixed and centrifuged for 5 min at 1763 g . (Glyco-)peptides were analyzed using an Ultimate 3000 RSLCnano system (Dionex/Thermo Fisher Scientific, Sunnyvale, CA) coupled to an Impact qTOF-MS (Bruker Daltonics, Bremen, Germany) as described previously.

    Techniques: Sequencing, Derivative Assay

    Accumulation of unique peptide spectral matches with replicate experiments. The number of unique peptides identified is shown with respect to the number of combined nanoLC-FAIMS-MS experiments at different compensation voltages (squares) or combined replicate

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Nanospray FAIMS Fractionation Provides Significant Increases in Proteome Coverage of Unfractionated Complex Protein Digests *

    doi: 10.1074/mcp.M111.014985

    Figure Lengend Snippet: Accumulation of unique peptide spectral matches with replicate experiments. The number of unique peptides identified is shown with respect to the number of combined nanoLC-FAIMS-MS experiments at different compensation voltages (squares) or combined replicate

    Article Snippet: Multiple analyses of 1 μg of SILAC-labeled yeast tryptic digest were performed with nanoLC-MS and nanoLC-FAIMS-MS. Forty replicate nanoLC-MS experiments were performed using Thermo Fisher's nanospray source, and 40 nanoLC-FAIMS-MS experiments were performed using the above-described modified HESI-II source.

    Techniques: Mass Spectrometry

    FAIMS increases peptide and protein identifications. Combining the results of all the nanoLC-FAIMS-MS experiments resulted in ( A ) a 64% increase in identification of proteins with SILAC ratios and ( B ) a 50% increase in identification of unique stripped

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Nanospray FAIMS Fractionation Provides Significant Increases in Proteome Coverage of Unfractionated Complex Protein Digests *

    doi: 10.1074/mcp.M111.014985

    Figure Lengend Snippet: FAIMS increases peptide and protein identifications. Combining the results of all the nanoLC-FAIMS-MS experiments resulted in ( A ) a 64% increase in identification of proteins with SILAC ratios and ( B ) a 50% increase in identification of unique stripped

    Article Snippet: Multiple analyses of 1 μg of SILAC-labeled yeast tryptic digest were performed with nanoLC-MS and nanoLC-FAIMS-MS. Forty replicate nanoLC-MS experiments were performed using Thermo Fisher's nanospray source, and 40 nanoLC-FAIMS-MS experiments were performed using the above-described modified HESI-II source.

    Techniques: Mass Spectrometry

    FAIMS increases dynamic range despite decreased signal intensity. The distribution of precursor ion intensities are shown for the combined nanoLC-FAIMS-MS experiments (solid line) and nanoLC-MS experiments (dashed line). The highest intensity precursor

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Nanospray FAIMS Fractionation Provides Significant Increases in Proteome Coverage of Unfractionated Complex Protein Digests *

    doi: 10.1074/mcp.M111.014985

    Figure Lengend Snippet: FAIMS increases dynamic range despite decreased signal intensity. The distribution of precursor ion intensities are shown for the combined nanoLC-FAIMS-MS experiments (solid line) and nanoLC-MS experiments (dashed line). The highest intensity precursor

    Article Snippet: Multiple analyses of 1 μg of SILAC-labeled yeast tryptic digest were performed with nanoLC-MS and nanoLC-FAIMS-MS. Forty replicate nanoLC-MS experiments were performed using Thermo Fisher's nanospray source, and 40 nanoLC-FAIMS-MS experiments were performed using the above-described modified HESI-II source.

    Techniques: Mass Spectrometry