rabbit anti human cxcl8  (Proteintech)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miRNA-503 inhibition exerts anticancer effects and reduces tumor growth in mesothelioma

doi: 10.1186/s13046-025-03283-0

Figure Lengend Snippet: Histological analysis of miR-503 target genes and transcriptomic analysis of miR-503 inhibition. A Evaluation of the expression of BTG1, CCNG1, EDG1 and TIMP2, in normal pleural (PL) and pleural mesothelioma tissues (EPM epithelial istotype; SPM, sacomatoid istotype). Yellow Scale bar = 100 μm. Data shown are mean ± S.D. of five independent experiments. B IHC analysis of the putative miR-503 target genes on mice tumors treated with LNPs-anti-miR-503 or empty LNPs (original magnification 20X). A and B confirm the in vitro results. C Transcriptome analysis in MSTO cells after miR-503 inhibition detected about 500 deregulated genes (red upregulated, green downregulated). D Expression analysis of MM biomarkers deregulated by inhibition of miR-503 in MM cells. E q-PCR analysis in human MM samples of the miR-503 MM biomarkers confirms the inverse expression only for CXCL8, SERPINE1 and SPP. F IHC analysis of the miR-503 deregulated MM biomarkers on mice tumors treated with LNPs-anti-miR-503 or empty LNPs (original magnification 20X). For each IHC panel bar plots report the score for the samples analysed. Data presented as the mean ± SD of at least three independent experiments ( n = 3) for the in vitro experiments while for the in vivo analysis numbers of samples are reported in Methods. * p < 0.05, ** p < 0.01 *** p < 0.001 **** p < 0.0001 vs. control

Article Snippet: The primary antibodies used were: Rabbit anti-human Ki67 (DAKO Agilent, Santa Clara, CA USA), Rabbit anti-human BTG1 (orb35408 Biorbyt, Durham, NC, USA), Rabbit anti-human CCNG1 (orb167206 Biorbyt), Rabbit anti-human S1PR1 (EDG1) (orb350684 Biorbyt), Rabbit anti-human TIMP2 (orb543218 Biorbyt), Rabbit anti-human CXCL8 (17038-1-AP Proteintech Rosemont, IL, USA), Rabbit anti-human Osteopontin, (SPP 20416-1-AP Proteintech), Rabbit anti-human SERPINE1 (PAI-1 #49536 Cell Signaling, Danvers, MA, USA).

Techniques: Inhibition, Expressing, In Vitro, In Vivo, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Flow chart representing the recruitment of LTx patients for analysis of the neutrophil phenotype (cohort 1) and CXCL8 proteoforms (cohort 2). ( A ) To study the neutrophil phenotype, a first cohort of 100 fresh BAL fluid and 50 peripheral blood samples were collected from 134 individual patients, including 16 patients with a paired BAL fluid and peripheral blood sample. BAL fluid samples were excluded for flow cytometry analysis if cell counts were insufficient or upon severe blood contamination. Samples with insufficient neutrophils for proper flow cytometry analysis were excluded as well. Other reasons for exclusion were samples derived from patients on post-operative day 1 or upon hospital discharge immediately after transplantation, from patients with other severe complications (anastomotic stricture, pulmonary embolisms) or without clear diagnosis. Samples were categorized retrospectively to be derived from LTx patients with CLAD or pulmonary infection or stable LTx recipients according to clinical guidelines (see section). ( B ) To study CXCL8 proteoforms in LTx, COVID-19 and influenza patients, -80 °C-stored BAL fluid supernatants from a second cohort of 55 individual patients were used

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Flow Cytometry, Derivative Assay, Transplantation Assay, Biomarker Discovery, Infection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Clinical characteristics CXCL8 proteoform cohort

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Infection, Biomarker Discovery, Cell Counting

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Detection of endogenous CXCL8 proteoforms in BAL fluid. ( A - C ) Neutrophil counts and CXCL8 levels in BAL fluid samples from CLAD ( n = 12), infected ( n = 6) and stable LTx patients ( n = 6) used for CXCL8 proteoform characterization. ( D , E ) Endogenous CXCL8 proteoforms were determined by ISTAMPA in the BAL fluid supernatants of LTx patients with CLAD or infection. Total CXCL8 levels were below the detection limit for ISTAMPA analysis in stable LTx recipients. CXCL8 proteoforms were presented according to their relative abundance in the BAL fluids, expressed as percentage of total CXCL8. ( F , G ) Correlation between the relative abundance of the most potent proteoform CXCL8(9–77) and the percentage and absolute numbers of neutrophils in the LTx BAL fluid samples (CLAD and infection combined). ( H , I ) ISTAMPA analysis of endogenous CXCL8 proteoforms in COVID-19 ( n = 24) and influenza ( n = 7) BAL fluid supernatants. Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; ** p < 0.01; *** p < 0.001. Correlation analysis was performed calculating a Spearman correlation coefficient and plotted utilizing a simple linear regression line

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Infection, Whisker Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Proteolytic processing of intact CXCL8 in BAL fluid. ( A ) Recombinant human CXCL8(1–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant from CLAD ( n = 12), infected ( n = 6) or stable LTx patients ( n = 6) and from patients with COVID-19 ( n = 10) or influenza ( n = 7). After incubation for 3 h at 37 °C, CXCL8 proteolysis was analyzed by ISTAMPA. ( B ) CXCL8 proteoforms were also determined immediately after spiking CXCL8 [without 37 °C incubation but including the required 30 min pre-purification of the CXCL8 proteoforms at room temperature (RT) as part of the ISTAMPA procedure] and after 16 h of incubation at 37 °C in the COVID-19 BAL fluids. CXCL8 proteoforms were presented according to their relative abundance in the BAL fluids, expressed as percentage of total CXCL8. ( C , D ) Correlation between the relative abundance of the intact CXCL8(1–77) or the truncated CXCL8(6–77) proteoform after 3 h incubation at 37 °C and the elastinolytic activity in the COVID-19 BAL fluid samples . Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; **** p < 0.0001. Correlation analysis was performed calculating a Spearman correlation coefficient and plotted utilizing a simple linear regression line

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Recombinant, Infection, Incubation, Purification, Activity Assay, Whisker Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Inhibition of proteolytic processing of CXCL8 in BAL fluid. ( A ) Recombinant human CXCL8(6–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant ( n = 3–6) from COVID-19 (indicated by dots) and CLAD patients (indicated by triangles) and incubated for 3 h at 37 °C, in the absence or presence of the metalloprotease inhibitor EDTA. ( B ) Recombinant human CXCL8(1–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant ( n = 8–9) from COVID-19 (dots) and CLAD patients (triangles) and incubated for 3 h at 37 °C, in the absence or presence of the serine protease inhibitor AEBSF or/and the metalloprotease inhibitor EDTA. After incubation, CXCL8 proteolysis was determined by ISTAMPA. All CXCL8 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL8. Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Mann-Whitney U tests or Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; *** p < 0.001; **** p < 0.0001

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Inhibition, Recombinant, Incubation, Protease Inhibitor, Whisker Assay, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Flow chart representing the recruitment of LTx patients for analysis of the neutrophil phenotype (cohort 1) and CXCL8 proteoforms (cohort 2). ( A ) To study the neutrophil phenotype, a first cohort of 100 fresh BAL fluid and 50 peripheral blood samples were collected from 134 individual patients, including 16 patients with a paired BAL fluid and peripheral blood sample. BAL fluid samples were excluded for flow cytometry analysis if cell counts were insufficient or upon severe blood contamination. Samples with insufficient neutrophils for proper flow cytometry analysis were excluded as well. Other reasons for exclusion were samples derived from patients on post-operative day 1 or upon hospital discharge immediately after transplantation, from patients with other severe complications (anastomotic stricture, pulmonary embolisms) or without clear diagnosis. Samples were categorized retrospectively to be derived from LTx patients with CLAD or pulmonary infection or stable LTx recipients according to clinical guidelines (see section). ( B ) To study CXCL8 proteoforms in LTx, COVID-19 and influenza patients, -80 °C-stored BAL fluid supernatants from a second cohort of 55 individual patients were used

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Flow Cytometry, Derivative Assay, Transplantation Assay, Biomarker Discovery, Infection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Clinical characteristics CXCL8 proteoform cohort

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Infection, Biomarker Discovery, Cell Counting

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Detection of endogenous CXCL8 proteoforms in BAL fluid. ( A - C ) Neutrophil counts and CXCL8 levels in BAL fluid samples from CLAD ( n = 12), infected ( n = 6) and stable LTx patients ( n = 6) used for CXCL8 proteoform characterization. ( D , E ) Endogenous CXCL8 proteoforms were determined by ISTAMPA in the BAL fluid supernatants of LTx patients with CLAD or infection. Total CXCL8 levels were below the detection limit for ISTAMPA analysis in stable LTx recipients. CXCL8 proteoforms were presented according to their relative abundance in the BAL fluids, expressed as percentage of total CXCL8. ( F , G ) Correlation between the relative abundance of the most potent proteoform CXCL8(9–77) and the percentage and absolute numbers of neutrophils in the LTx BAL fluid samples (CLAD and infection combined). ( H , I ) ISTAMPA analysis of endogenous CXCL8 proteoforms in COVID-19 ( n = 24) and influenza ( n = 7) BAL fluid supernatants. Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; ** p < 0.01; *** p < 0.001. Correlation analysis was performed calculating a Spearman correlation coefficient and plotted utilizing a simple linear regression line

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Infection, Whisker Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Proteolytic processing of intact CXCL8 in BAL fluid. ( A ) Recombinant human CXCL8(1–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant from CLAD ( n = 12), infected ( n = 6) or stable LTx patients ( n = 6) and from patients with COVID-19 ( n = 10) or influenza ( n = 7). After incubation for 3 h at 37 °C, CXCL8 proteolysis was analyzed by ISTAMPA. ( B ) CXCL8 proteoforms were also determined immediately after spiking CXCL8 [without 37 °C incubation but including the required 30 min pre-purification of the CXCL8 proteoforms at room temperature (RT) as part of the ISTAMPA procedure] and after 16 h of incubation at 37 °C in the COVID-19 BAL fluids. CXCL8 proteoforms were presented according to their relative abundance in the BAL fluids, expressed as percentage of total CXCL8. ( C , D ) Correlation between the relative abundance of the intact CXCL8(1–77) or the truncated CXCL8(6–77) proteoform after 3 h incubation at 37 °C and the elastinolytic activity in the COVID-19 BAL fluid samples . Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; **** p < 0.0001. Correlation analysis was performed calculating a Spearman correlation coefficient and plotted utilizing a simple linear regression line

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Recombinant, Infection, Incubation, Purification, Activity Assay, Whisker Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Inhibition of proteolytic processing of CXCL8 in BAL fluid. ( A ) Recombinant human CXCL8(6–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant ( n = 3–6) from COVID-19 (indicated by dots) and CLAD patients (indicated by triangles) and incubated for 3 h at 37 °C, in the absence or presence of the metalloprotease inhibitor EDTA. ( B ) Recombinant human CXCL8(1–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant ( n = 8–9) from COVID-19 (dots) and CLAD patients (triangles) and incubated for 3 h at 37 °C, in the absence or presence of the serine protease inhibitor AEBSF or/and the metalloprotease inhibitor EDTA. After incubation, CXCL8 proteolysis was determined by ISTAMPA. All CXCL8 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL8. Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Mann-Whitney U tests or Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; *** p < 0.001; **** p < 0.0001

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Inhibition, Recombinant, Incubation, Protease Inhibitor, Whisker Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Workflow for immunosorbent tandem mass spectrometry proteoform analysis (ISTAMPA). (1) Synovial fluids are collected from the inflamed joints of patients with arthritis. (2) Total CXCL8 is extracted from patient samples by immunosorbent purification using antibody-coupled magnetic beads. (3) Isolated CXCL8 proteoforms are subjected to analysis by nano-UPLC-MS/MS. (4, 5) The detection and quantification of signature fragment ions, generated by top-down collision-induced dissociation (CID) of a pre-selected precursor ion, at the expected elution time point, strongly indicates the presence of a specific CXCL8 proteoform. In general, truncated CXCL8 proteoforms display an enhanced capacity to induce neutrophil migration and activation. This figure is created with BioRender software.

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Mass Spectrometry, Purification, Magnetic Beads, Isolation, Tandem Mass Spectroscopy, Generated, Migration, Activation Assay, Software

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 proteoforms by top-down tandem mass spectrometry. (A) Parameters are optimized to ensure that only the acid-labile Asp-Pro (DP) bond breaks during low-energy fragmentation and only two fragment ions are generated. (B) Mass spectra (single MS) showing the intensity of multiple charged ions of CXCL8(1-77), CXCL8(6-77) and CXCL8(9-77) as a function of their m/z values. Ions with m/z values of 892.8 [for CXCL8(1-77)], 932.3 [for CXCL8(6-77)] or 900.4 [for CXCL8(9-77)] were isolated and fragmented with low energy CID. The resulting fragmentation spectra are shown in the lower panels in (C) . Diamonds indicate the m/z value of the precursor ions selected for fragmentation. Extracted ion chromatograms (EIC) (top panels) show the detection of m/z values (±0.5) of the two signature fragment ions during protein elution. Relative quantification of CXCL8 forms is performed based on the intensity of the two major fragment ions in the EIC. (D) Dose-response curves for the simultaneous quantification of three CXCL8 forms in MRM mode (amounts ranging from 3.1 pg to 12.5 ng). Regression analysis was performed to fit curves to data. Results are represented as mean ± SEM ( n ≥ 4).

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Mass Spectrometry, Generated, Isolation, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Site-specific fragmentation of CXCL8 proteoform ions by top-down ion trap tandem mass spectrometry at limited fragmentation energy.

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Mass Spectrometry, Sequencing

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 proteoforms in cell culture supernatant from osteosarcoma cells. The MG-63 osteosarcoma cell line was stimulated with the synthetic double stranded RNA poly rI:rC to produce CXCL8. Partially purified cell culture supernatant was subjected to top-down nano-LC-MS/MS analysis. Two nano-LC-MS/MS runs were performed to examine the presence and quantity of eight CXCL8 proteoforms in MRM mode. (A) Extracted ion chromatograms (EIC) showing the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. (B) Fragmentation spectra confirming the presence of signature ions of CXCL8(-2-77), CXCL8(1-77), CXCL8(3-77), CXCL8(6-77), CXCL8(7-77) and CXCL8(8-77). (C) Relative abundance of the detected CXCL8 proteoforms.

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, Purification, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 by top down-tandem mass spectrometry proves proteolytic activation in synovial fluids of arthritis patients. Total CXCL8 was extracted from synovial fluids of RA ( n = 14) and JIA patients ( n = 12) by immunosorbent isolation and subjected to nano-LC-MS/MS analysis. For each sample, two top-down nano-LC-MS/MS runs were performed to examine the presence of eight CXCL8 forms in MRM mode. (A) Extracted ion chromatograms (EIC) show the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. A representative experiment is shown (RA patient). (B) Representative fragmentation spectra confirm the presence of signature ions of CXCL8(1-77), CXCL8(6-77), CXCL8(7-77), CXCL8(8-77) and CXCL8(9-77) in synovial fluid from a patient with RA. (C) Relative abundance of CXCL8 proteoforms in synovial fluids from RA and JIA patients (represented as mean ± SEM).

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Mass Spectrometry, Activation Assay, Isolation, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Kinetics of CXCL8 processing in the presence of synovial fluids from juvenile idiopathic arthritis patients. Exogenous CXCL8(1-77) was incubated with synovial fluids from JIA patients ( n = 4) for a period of 0, 1, 3, 6, 12 or 20 h. Total CXCL8 was extracted from synovial fluids by immunosorbent isolation and subjected to nano-LC-MS/MS analysis. For each sample, two nano-LC-MS/MS runs were performed to examine the presence of eight CXCL8 forms in MRM mode. (A) Extracted ion chromatograms (EIC) show the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. (B) Relative abundance of CXCL8 proteoforms in synovial fluids after 0, 1, 3, 6, 12, and 20 h of incubation (represented as mean ± SEM). (C) Pseudo-first order association kinetics of proteolytic modification of CXCL8(1-77) in the presence of synovial fluids (represented as mean ± SEM; n = 4).

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Incubation, Isolation, Liquid Chromatography with Mass Spectroscopy, Generated, Modification

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Workflow for immunosorbent tandem mass spectrometry proteoform analysis (ISTAMPA). (1) Synovial fluids are collected from the inflamed joints of patients with arthritis. (2) Total CXCL8 is extracted from patient samples by immunosorbent purification using antibody-coupled magnetic beads. (3) Isolated CXCL8 proteoforms are subjected to analysis by nano-UPLC-MS/MS. (4, 5) The detection and quantification of signature fragment ions, generated by top-down collision-induced dissociation (CID) of a pre-selected precursor ion, at the expected elution time point, strongly indicates the presence of a specific CXCL8 proteoform. In general, truncated CXCL8 proteoforms display an enhanced capacity to induce neutrophil migration and activation. This figure is created with BioRender software.

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Mass Spectrometry, Purification, Magnetic Beads, Isolation, Tandem Mass Spectroscopy, Generated, Migration, Activation Assay, Software

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 proteoforms by top-down tandem mass spectrometry. (A) Parameters are optimized to ensure that only the acid-labile Asp-Pro (DP) bond breaks during low-energy fragmentation and only two fragment ions are generated. (B) Mass spectra (single MS) showing the intensity of multiple charged ions of CXCL8(1-77), CXCL8(6-77) and CXCL8(9-77) as a function of their m/z values. Ions with m/z values of 892.8 [for CXCL8(1-77)], 932.3 [for CXCL8(6-77)] or 900.4 [for CXCL8(9-77)] were isolated and fragmented with low energy CID. The resulting fragmentation spectra are shown in the lower panels in (C) . Diamonds indicate the m/z value of the precursor ions selected for fragmentation. Extracted ion chromatograms (EIC) (top panels) show the detection of m/z values (±0.5) of the two signature fragment ions during protein elution. Relative quantification of CXCL8 forms is performed based on the intensity of the two major fragment ions in the EIC. (D) Dose-response curves for the simultaneous quantification of three CXCL8 forms in MRM mode (amounts ranging from 3.1 pg to 12.5 ng). Regression analysis was performed to fit curves to data. Results are represented as mean ± SEM ( n ≥ 4).

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Mass Spectrometry, Generated, Isolation

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Site-specific fragmentation of CXCL8 proteoform ions by top-down ion trap tandem mass spectrometry at limited fragmentation energy.

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Mass Spectrometry, Sequencing

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 proteoforms in cell culture supernatant from osteosarcoma cells. The MG-63 osteosarcoma cell line was stimulated with the synthetic double stranded RNA poly rI:rC to produce CXCL8. Partially purified cell culture supernatant was subjected to top-down nano-LC-MS/MS analysis. Two nano-LC-MS/MS runs were performed to examine the presence and quantity of eight CXCL8 proteoforms in MRM mode. (A) Extracted ion chromatograms (EIC) showing the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. (B) Fragmentation spectra confirming the presence of signature ions of CXCL8(-2-77), CXCL8(1-77), CXCL8(3-77), CXCL8(6-77), CXCL8(7-77) and CXCL8(8-77). (C) Relative abundance of the detected CXCL8 proteoforms.

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Cell Culture, Purification, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 by top down-tandem mass spectrometry proves proteolytic activation in synovial fluids of arthritis patients. Total CXCL8 was extracted from synovial fluids of RA ( n = 14) and JIA patients ( n = 12) by immunosorbent isolation and subjected to nano-LC-MS/MS analysis. For each sample, two top-down nano-LC-MS/MS runs were performed to examine the presence of eight CXCL8 forms in MRM mode. (A) Extracted ion chromatograms (EIC) show the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. A representative experiment is shown (RA patient). (B) Representative fragmentation spectra confirm the presence of signature ions of CXCL8(1-77), CXCL8(6-77), CXCL8(7-77), CXCL8(8-77) and CXCL8(9-77) in synovial fluid from a patient with RA. (C) Relative abundance of CXCL8 proteoforms in synovial fluids from RA and JIA patients (represented as mean ± SEM).

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Mass Spectrometry, Activation Assay, Isolation, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Kinetics of CXCL8 processing in the presence of synovial fluids from juvenile idiopathic arthritis patients. Exogenous CXCL8(1-77) was incubated with synovial fluids from JIA patients ( n = 4) for a period of 0, 1, 3, 6, 12 or 20 h. Total CXCL8 was extracted from synovial fluids by immunosorbent isolation and subjected to nano-LC-MS/MS analysis. For each sample, two nano-LC-MS/MS runs were performed to examine the presence of eight CXCL8 forms in MRM mode. (A) Extracted ion chromatograms (EIC) show the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. (B) Relative abundance of CXCL8 proteoforms in synovial fluids after 0, 1, 3, 6, 12, and 20 h of incubation (represented as mean ± SEM). (C) Pseudo-first order association kinetics of proteolytic modification of CXCL8(1-77) in the presence of synovial fluids (represented as mean ± SEM; n = 4).

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Incubation, Isolation, Liquid Chromatography with Mass Spectroscopy, Generated, Modification