Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome *

doi: 10.1074/mcp.RA118.001304

Figure Lengend Snippet: KLK7 directly activates pro-MMP10. A, Peptograph (replicate 2) representing KLK7-mediated hydrolysis of pro-MMP10 in SKOV-3 cell CM. See supplemental Fig. S2 for peptograph description. The higher molecular weight (MW) peptides (blue - Heavy labeled) in gel slice 7 (Log2 KLK7/control = −3 to −5) represent the full length pro-MMP10 identified in the buffer-treated sample. The lower MW fragments in gel slices 11 and 20 (Log2 KLK7/control = 3 to 5) represent the KLK7 cleavage fragments identified in the KLK7-treated sample (red - Light labeled). A schematic of selected protein domains, based on annotation in the UniProtKB, is shown beneath the X-axis (purple boxes), aligned with the appropriate residues. Pro, activation peptide; PEX, hemopexin domain; the molecular weight of the protein standard (kDa) is indicated to the left. Arrow heads in colors depict, open: pro-MMP10 full length; black, KLK7-generated MMP10 fragments. The box plot in the middle panel represents the distribution of ratios found in each gel slice. B, Spectrum for the TAILS identified KLK7 cleavage site C-terminal to F99 and is also shown beneath the domain structure in a red dotted vertical line in A. C, Peptides identified in the qPROTOMAP analysis in gel slices 6, 7, 11 and 20 (PEP, posterior error probability; Score, the sum of the ion scores of all peptides identified; PSMs, peptide spectrum matches) with their respective Light/Heavy ratio, count and Log2 value. D, Silver-stained 12% SDS-PAGE showing hydrolysis of recombinant (r) pro-MMP10 (400 ng) by recombinant active KLK7 (1/10 −1/1000 molar ratio to rpro-MMP10); buffer and dmKLK7 treatments were used as controls. Arrow heads in colors depict, open: pro-MMP10 full length protein; red: 45 kDa cleavage fragment; yellow: cleavage fragment ∼22 and 30 kDa; black: KLK7 or dmKLK7. E, Pro-MMP10 treated with KLK7 (black squares) showed increasing fluorescence emission over time compared with the controls. Pro-MMP10 (yellowish brown circles) showed a slight increase in fluorescence emission with the fluorogenic peptide substrate over time, indicating partial activity, because of residual active MMP10 in the recombinant pro-MMP10 protein purchased. KLK7 (red diamonds) or dmKLK7 (green circles) did not show activity with the MMP10 fluorogenic peptide substrate, confirming that fluorescence intensity increases in the KLK-treated pro-MMP10 is because of putative KLK7-activated-pro-MMP10, but not because of residual KLK7 activity. F, Bar graph represents end point (at 35 min) of the reactions performed with triplicate biological replicates (n = 3) with the error bar representing the standard error of the mean. In all instances blank corrected fluorescence values were plotted.

Article Snippet: ATCC American Type Culture Collection dmKLK7 double mutant Kallikreinrelated peptidase 7 BCA bicinchoninic acid assay BSA bovine serum albumin CM conditioned media COL collagen DPBS Dulbecco's phosphate-buffered saline ECM extracellular matrix FBS fetal bovine serum FDR false discovery rate HPG-ALD hyper branched polyglycerol-aldehyde IAA iodoacetamide IDA Information Dependent Acquisition IGFBP insulin-like growth factor binding protein IPA ingenuity pathway analysis MMP matrix metalloprotease qPROTOMAP quantitative PROtein TOpography and Migration Analysis Platform SILAC stable isotopic labelling by amino acids in cell culture TAILS Terminal Amine Isotopic Labelling of Substrates TPP trans proteomics pipeline THBS1 thrombospondin 1 UniProtKB UniProt Knowledgebase.

Techniques: Molecular Weight, Labeling, Control, Activation Assay, Generated, Staining, SDS Page, Recombinant, Fluorescence, Activity Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome *

doi: 10.1074/mcp.RA118.001304

Figure Lengend Snippet: KLK7-mediated cleavage of thrombospondin 1. A, Peptograph (replicate 1) representing KLK7-mediated hydrolysis of THBS1 in SKOV-3 cell CM See supplemental Fig. S2 for peptograph description. Arrowheads to the right indicate the migration of THBS1 retrieved from the control (open) and KLK7-generated fragments of THBS1 (filled). The higher molecular weight (MW) peptides (blue and gray) are abundant in gel slices 1–4 (Log2 KLK7/control = 0 to −5), representing the fragments derived from the full-length protein identified in both KLK7- and buffer-treated samples. The lower MW fragments are abundant in gel slices 5–15 (Log2 KLK7/control = 0–5), representing the KLK7 cleavage fragments (red) found in the KLK7-treated sample. A schematic of selected protein domains, based on annotation in the UniProtKB, is shown beneath the X-axis (purple boxes), aligned with the appropriate residues. H, heparin-binding; V, von Willebrand factor, type-C; THBS1/3, THBS type-1/3 repeat; E, epidermal growth factor-like; THBS C-, THBS C-terminal. The TAILS identified KLK7 cleavage sites C-terminal to Y258 and Y665 are shown by red dotted vertical lines and B, represents the respective spectrums. C, Peptides identified in the qPROTOMAP analysis (PEP, posterior error probability; Score, The sum of the ion scores of all peptides identified; PSMs, peptide spectrum matches). D, Silver-stained 12% SDS-PAGE showing hydrolysis of recombinant (r) THBS1 (500 ng) by recombinant active KLK7 (1/10 −1/1000 molar ratio to rTHBS1); buffer and dmKLK7 treatments were used as controls. Arrow heads in colors depict, yellow: THBS1 full length protein; white: cleavage fragment ∼130 kDa; red: 28 kDa cleavage fragment; black: KLK7 or dmKLK7. Western blot analysis of KLK7-treated rTHBS1 using antibodies targeting E, full length and F, N-terminal THBS1 confirmed the KLK7-mediated generation of N-terminal heparin binding domain containing fragment. Arrowheads indicate different protein products: yellow: full length THBS1, 150 kDa; white: 130 kDa; green: 100 kDa; gray: 90 kDa; red: 28 and 25 kDa fragments. The MW of the protein standard (kDa) is indicated to the left.

Article Snippet: ATCC American Type Culture Collection dmKLK7 double mutant Kallikreinrelated peptidase 7 BCA bicinchoninic acid assay BSA bovine serum albumin CM conditioned media COL collagen DPBS Dulbecco's phosphate-buffered saline ECM extracellular matrix FBS fetal bovine serum FDR false discovery rate HPG-ALD hyper branched polyglycerol-aldehyde IAA iodoacetamide IDA Information Dependent Acquisition IGFBP insulin-like growth factor binding protein IPA ingenuity pathway analysis MMP matrix metalloprotease qPROTOMAP quantitative PROtein TOpography and Migration Analysis Platform SILAC stable isotopic labelling by amino acids in cell culture TAILS Terminal Amine Isotopic Labelling of Substrates TPP trans proteomics pipeline THBS1 thrombospondin 1 UniProtKB UniProt Knowledgebase.

Techniques: Migration, Control, Generated, Molecular Weight, Derivative Assay, Binding Assay, Staining, SDS Page, Recombinant, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome *

doi: 10.1074/mcp.RA118.001304

Figure Lengend Snippet:

Article Snippet: ATCC American Type Culture Collection dmKLK7 double mutant Kallikreinrelated peptidase 7 BCA bicinchoninic acid assay BSA bovine serum albumin CM conditioned media COL collagen DPBS Dulbecco's phosphate-buffered saline ECM extracellular matrix FBS fetal bovine serum FDR false discovery rate HPG-ALD hyper branched polyglycerol-aldehyde IAA iodoacetamide IDA Information Dependent Acquisition IGFBP insulin-like growth factor binding protein IPA ingenuity pathway analysis MMP matrix metalloprotease qPROTOMAP quantitative PROtein TOpography and Migration Analysis Platform SILAC stable isotopic labelling by amino acids in cell culture TAILS Terminal Amine Isotopic Labelling of Substrates TPP trans proteomics pipeline THBS1 thrombospondin 1 UniProtKB UniProt Knowledgebase.

Techniques: