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