human recombinant cims scfv antibody fragments  (BioInvent)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Targeting Classes of Motif-containing Peptides Using Immunoaffinity-based Mass Spectrometry *

doi: 10.1074/mcp.M111.016238

Figure Lengend Snippet: Quantitative accuracy and reproducibility. A , Experimentally refined binding motif amino acid sequences as compiled from all MS-MS detected captured peptides (excluding background peptides) in the spike-in proteome experiments (glucose grown SILAC-labeled yeast proteomes mixed at three ratios of H and L) for two CIMS antibodies, clones CIMS17-E02 (97 peptides) and CIMS-33–3D-F06 (142 peptides). The frequency of each individual residue in the last six C-terminal positions is indicated. B , Peptide intensity/ratio distribution for peptides enriched, from predefined proteome mixtures, by the two CIMS antibodies. Data limited to YXR motif-containing peptides for CIMS-17-E02 and DXR motif-containing peptides for CIMS-33–3D-F06. (The two replicate captures for each condition were analyzed as one experiment in MaxQuant. Hence, raw (non-normalized) ratios, as calculated from associated redundant peptide ratios by means of median of the two replicates, are displayed. Furthermore, data limited to peptides with MaxQuant reported ratios in all conditions and replicate capture trials resulting in 39 different peptides for CIMS-17-E02 and 64 different peptides for CIMS-33–3D-F06). Noteworthy, the peptides were ordered based on total intensity, resulting in that peptide number 39 and 64 were the two peptides observed with the highest total intensity. C , The same data as in panel B , but now compensated (normalized) against the corresponding 50/50 ratio measurement set to be 0. D , Reproducibility between replicate capture experiments for CIMS-17-E02 (limited to YXR motif-containing peptides) and CIMS-33–3D-F06 (limited to DXR motif-containing peptides).

Article Snippet: Six human recombinant CIMS scFv antibodies (clones 1-B03, 15-A06, 17-E02, 32–3A-G03, 33–3D-F06, and 34–3A-D10) directed against six short C-terminal amino acid peptide motifs (denoted M-1, M-15, M17, M-32, M-33, and M-34), were selected from the n-CoDeR library , and kindly provided by BioInvent International AB, Lund, Sweden ( supplemental Table S1 ).

Techniques: Binding Assay, Tandem Mass Spectroscopy, Multiplex sample analysis, Labeling, Clone Assay, Residue

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Targeting Classes of Motif-containing Peptides Using Immunoaffinity-based Mass Spectrometry *

doi: 10.1074/mcp.M111.016238

Figure Lengend Snippet: Dynamic range and sensitivity. Glucose grown yeast SILAC-proteomes, mixed at three ratios of H and L, were profiled using two CIMS antibodies, A , CIMS-17-E02 and B , CIMS-33–3D-F06, and mapped to the known protein abundances generated by orthogonal methods . In total, the data was based on six capture trials (two for each isotopically labeled spike in proteome ratio mix) per CIMS-antibody ( supplemental Tables S3–S5 ). For Swiss-Prot ID's matching two different synonymous yeast SGD ids and two different values in , the highest reported value was plotted. In cases with multiple Swiss-Prot ID's, they were checked in alphabetical order for presence in , and if no value was present the second Swiss-Prot id was checked and onwards. A number of identified peptides/proteins were reported to be present in less than 50 copies/cell or had no reported value at all and thereby could not be plotted and are listed separately in a box.

Article Snippet: Six human recombinant CIMS scFv antibodies (clones 1-B03, 15-A06, 17-E02, 32–3A-G03, 33–3D-F06, and 34–3A-D10) directed against six short C-terminal amino acid peptide motifs (denoted M-1, M-15, M17, M-32, M-33, and M-34), were selected from the n-CoDeR library , and kindly provided by BioInvent International AB, Lund, Sweden ( supplemental Table S1 ).

Techniques: Multiplex sample analysis, Generated, Labeling

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Targeting Classes of Motif-containing Peptides Using Immunoaffinity-based Mass Spectrometry *

doi: 10.1074/mcp.M111.016238

Figure Lengend Snippet: Coverage—comparison of GPS and SCX. Yeast SILAC-proteomes, cultivated in ethanol or glucose, were profiled using GPS and SCX. A , Venn diagram showing the number of peptides identified by GPS (based on 6 CIMS antibodies) and SCX; a total number of 4126 peptides were identified of which 417 were detected in both. B , Venn diagram showing the number of proteins identified by GPS and SCX; a total number of 987 proteins were identified of which 382 were detected in both. C , Statistics of all the identified peptides and proteins. D , peptide length plotted for all identified peptides using GPS and SCX. E , peptide length plotted for peptides identified in both biological replicates with a ratio count of ≥2 using GPS and SCX.

Article Snippet: Six human recombinant CIMS scFv antibodies (clones 1-B03, 15-A06, 17-E02, 32–3A-G03, 33–3D-F06, and 34–3A-D10) directed against six short C-terminal amino acid peptide motifs (denoted M-1, M-15, M17, M-32, M-33, and M-34), were selected from the n-CoDeR library , and kindly provided by BioInvent International AB, Lund, Sweden ( supplemental Table S1 ).

Techniques: Comparison, Multiplex sample analysis

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Targeting Classes of Motif-containing Peptides Using Immunoaffinity-based Mass Spectrometry *

doi: 10.1074/mcp.M111.016238

Figure Lengend Snippet: Quantitative data—comparison of GPS and SCX. SILAC-labeled yeast proteomes, cultivated in ethanol or glucose, were profiled using GPS (based on six CIMS antibodies) and SCX. The correlation between the data sets was examined. A , all identified peptides for biosample 1 (isotopic amino acids present in ethanol condition). B , normalized data for all identified peptides in biosample 1 (isotopic amino acids present in ethanol condition). C , all identified proteins for biosample 1 (isotopic amino acids present in ethanol condition). D , normalized data for all identified proteins in biosample 1 (isotopic amino acids present in ethanol condition).

Article Snippet: Six human recombinant CIMS scFv antibodies (clones 1-B03, 15-A06, 17-E02, 32–3A-G03, 33–3D-F06, and 34–3A-D10) directed against six short C-terminal amino acid peptide motifs (denoted M-1, M-15, M17, M-32, M-33, and M-34), were selected from the n-CoDeR library , and kindly provided by BioInvent International AB, Lund, Sweden ( supplemental Table S1 ).

Techniques: Comparison, Multiplex sample analysis, Labeling

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Targeting Classes of Motif-containing Peptides Using Immunoaffinity-based Mass Spectrometry *

doi: 10.1074/mcp.M111.016238

Figure Lengend Snippet: Identification repeatability—comparison of GPS and SCX. The reproducibility of the MS/MS identification was evaluated by determining the identification overlap for all replicate runs. A , percentage peptide ID overlap in replicate technical (capture + LC-MS/MS) and biological replicate GPS-assays (based on six CIMS antibodies) (biosample 1, ethanol (K 6 , R 10 ) - glucose; biosample 2, ethanol - glucose (K 6 , R 10 )). It should be noted that the generated values were based on separate capture and LC-MS/MS steps. B , percentage peptide ID overlap for replicate runs (same SCX-fraction analyzed twice with LC-MS/MS) and biological replicate runs for the eight generated SCX fractions.

Article Snippet: Six human recombinant CIMS scFv antibodies (clones 1-B03, 15-A06, 17-E02, 32–3A-G03, 33–3D-F06, and 34–3A-D10) directed against six short C-terminal amino acid peptide motifs (denoted M-1, M-15, M17, M-32, M-33, and M-34), were selected from the n-CoDeR library , and kindly provided by BioInvent International AB, Lund, Sweden ( supplemental Table S1 ).

Techniques: Comparison, Tandem Mass Spectroscopy, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Targeting Classes of Motif-containing Peptides Using Immunoaffinity-based Mass Spectrometry *

doi: 10.1074/mcp.M111.016238

Figure Lengend Snippet: Biological relevance of identified proteins for carbon and ethanol metabolism. The biological relevance of differentially expressed proteins in glucose versus ethanol yeast was evaluated. The GPS assay was based on six CIMS antibodies. A , all identified proteins by GPS for biosample 1 (ethanol (K 6 , R 10 ) - glucose) plotted as overall fold change as a function of protein intensity in the MS. Protein ratios are color-coded according to their ratio significance (significance B, as described in ). B , all identified proteins by SCX for biosample 1(ethanol (K 6 , R 10 ) - glucose) plotted as overall fold change as a function of protein intensity in the MS. Protein ratios are color-coded according to their ratio significance (significance B, as described in ). C , GPS and SCX analysis of the central carbon metabolism. Protein expression values generated by GPS and SCX were mapped to the central carbon metabolism pathway(s) displayed in a condensed version according to . D , Protein-protein interaction maps of GPS identified up- and down-regulated proteins for biosample 1. Thirty-one up-regulated proteins (limited to proteins reported/annotated with a single Swiss-Prot ID, MaxQuant significance B value of p < 0.05 resulting in proteins with ≥5.5-fold up-regulation). Twenty-three down-regulated proteins (limited to proteins reported/annotated with a single Swiss-Prot ID, MaxQuant significance B value of p < 0.1 resulting in proteins with ≥twofold down-regulation).

Article Snippet: Six human recombinant CIMS scFv antibodies (clones 1-B03, 15-A06, 17-E02, 32–3A-G03, 33–3D-F06, and 34–3A-D10) directed against six short C-terminal amino acid peptide motifs (denoted M-1, M-15, M17, M-32, M-33, and M-34), were selected from the n-CoDeR library , and kindly provided by BioInvent International AB, Lund, Sweden ( supplemental Table S1 ).

Techniques: Expressing, Generated

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis and Discovery Using Affinity Proteomics and Mass Spectrometry *

doi: 10.1074/mcp.M110.003962

Figure Lengend Snippet: Binding patterns of 14 CIMS antibodies against full-length semidenatured human proteins as determined by protein array screening

Article Snippet: Purified CIMS antibodies were individually coupled to POROS 20 AL bead (Applied Biosystems, Foster City, CA) using standard protocols.

Techniques: Binding Assay, Protein Array

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis and Discovery Using Affinity Proteomics and Mass Spectrometry *

doi: 10.1074/mcp.M110.003962

Figure Lengend Snippet: Structural analysis of two CIMS antibodies, denoted sister clones, and selected against the same selection motif. A ,Experimentally refined binding motif amino acid sequences as compiled from all MS/MS detected captured peptides, and the frequency of each individual residue in the last six C-terminal positions is indicated. The two sister clones were selected against the same original selection peptide motif, M-1 EDFR. B , Comparison of the amino acid sequence of the complementarity determining regions (CDRs) of the two sister clones. C , Three-dimensional homology models, shown as a ribbon structure (top view) and with color coded CDRs of the two sister clones. D , Space filling models of the three-dimensional structures shown in C. E , Space filling models of the structures shown in C , onto which the electrostatics have been mapped.

Article Snippet: Purified CIMS antibodies were individually coupled to POROS 20 AL bead (Applied Biosystems, Foster City, CA) using standard protocols.

Techniques: Clone Assay, Selection, Binding Assay, Tandem Mass Spectroscopy, Sequencing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis and Discovery Using Affinity Proteomics and Mass Spectrometry *

doi: 10.1074/mcp.M110.003962

Figure Lengend Snippet: First prototype GPS set-up targeting synthetic peptide mixtures or tryptic digests of complex proteomes. A , MALDI-TOF MS analysis of a mixture of 10 synthetic selection peptide motifs (mix-1 and 2), applied to micro-columns containing immobilized CIMS-17-E02 or CIMS-33–3D-F06 antibodies. B , MALDI-MS/MS analysis of tryptic digests from crude mouse liver homogenates applied to micro-columns containing immobilized CIMS-17-E02 or CIMS-33–3D-F06 antibodies. C , Statistics of the number of bound mouse peptides/proteins per CIMS antibody, as determined in B , and illustrated for four different CIMS antibodies. D , LC-MS/MS analysis of tryptic digests from crude human colon tissue applied to micro-columns or beads containing immobilized CIMS-17-E02 or CIMS-33–3D-F06 antibodies. E , Statistics of the number of bound human peptides/proteins per CIMS antibody, as determined in D , and illustrated for seven different CIMS antibodies. F , LC-MS/MS analysis of tryptic digests from crude yeast cell lysates applied to beads containing immobilized CIMS-17-E02 or CIMS-33–3D-F06 antibodies. G , Statistics of the number of bound yeast peptides/proteins per CIMS antibody, as determined in F , and illustrated for six different CIMS antibodies.

Article Snippet: Purified CIMS antibodies were individually coupled to POROS 20 AL bead (Applied Biosystems, Foster City, CA) using standard protocols.

Techniques: Selection, Tandem Mass Spectroscopy, Liquid Chromatography with Mass Spectroscopy

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis and Discovery Using Affinity Proteomics and Mass Spectrometry *

doi: 10.1074/mcp.M110.003962

Figure Lengend Snippet: Characterization of the GPS set-up. A , Multiplexing of GPS, as illustrated for two CIMS antibodies, CIMS-17-E02 and CIMS-33–3D-F06, which were mixed and subsequently exposed to tryptic digests from human colon tissue or yeast cell lysates, respectively and then analyzed by LC-MS/MS. The statistics of the number of bound human or yeast peptides or proteins per CIMS antibody are shown. B , Peptide identification reproducibility of the complete GPS-setup, including both affinity capture and LC-MS/MS as illustrated for CIMS-33–3D-F06 exposed to human tryptic colon digest or yeast cell lysates. All peptides identified with fixed or variable modifications were excluded. C , Reproducibility of the entire GPS setup for label-free quantitative LC-MS/MS, as illustrated for CIMS-33–3D-F06 exposed to different amounts of human tryptic colon digests or yeast cell lysates. Raw abundance values (nonnormalized area under the peak values generated by Progenesis LC-MS software) are plotted. Data plotted were limited to peptides containing the experimentally refined binding motif (the frequency of the last six C-terminal amino acids is indicated for the peptides plotted). D , Dynamic range of the GPS set-up, illustrated for six CIMS antibodies immobilized on magnetic beads and exposed to tryptic digest of yeast cell lysates. The identified peptides (proteins) were plotted against the á priori known protein abundance levels of all individual yeast proteins . Among the detected peptides, four are indicated for two CIMS antibodies each, CIMS-17-E02 and CIMS-33–3D-F06, demonstrating the dynamic coverage. Eight detected peptides for which reference values are missing (not plotted) and for which the abundance is anticipated to be <50 protein copies/cell are shown.

Article Snippet: Purified CIMS antibodies were individually coupled to POROS 20 AL bead (Applied Biosystems, Foster City, CA) using standard protocols.

Techniques: Multiplexing, Liquid Chromatography with Mass Spectroscopy, Generated, Software, Binding Assay, Magnetic Beads