panorama0000ProteomicsAlok ShahHomo Sapienshttps://panoramaweb.org/CytokineInSecretome.urlalok.shah@qimrberghofer.edu.auQIMR Berghofer Medical Research Institute, Brisbane, AustraliaPanoramaPublicCytokines are commonly measured by immunoassays; however, these have limited multiplexing capacity, are costly, and can exhibit cross-reactivity. Multiple reaction monitoring (MRM) mass spectrometry is a robust method to quantify analytes with high specificity and multiplexing ability, hence we aimed to investigate its suitability as an alternative cost-effective method for cytokine measurement. Human keratinocyte conditioned media spiked with recombinant cytokines was used as an experimental system to evaluate sensitivity, linearity, and reproducibility of an MRM assay targeting 79 peptides representing 23 human cytokines. Our MRM method was able to identify 21 cytokines by two or more unique peptides and two cytokines by a single unique peptide. In a serum-free matrix, the median LOD and LOQ for cytokine peptides was 130 and 433 pg/mL, respectively. The presence of serum increased median LOD and LOQ by about 2.3-fold. The assay shows excellent replicate consistency with 8% intra- and 12% interday coefficient of variations. We found high pH reversed-phase fractionation a useful tool to increase assay sensitivity with the drawback of increasing its variability by approximately 10%. Overall, our results suggest utility of a multiplex cytokine MRM for routine measurement of secreted cytokines in cellular experiments under low serum conditions. Additional enrichment steps will be required in high complexity matrices such as serum.Antibody-Free Multiplex Measurement of 23 Human Cytokines in Primary Cell Culture Secretome Using Targeted Mass Spectrometry.Krueger Annika A, Stoll Thomas T, Shah Alok K AK, Sinha Rohit R, Frazer Ian H IH, Hill Michelle M MMauthor summary, Biological Markers, Viral Marker, Inflammations, Surrogate Endpoints, PhrB photolyase activity, determination, Laboratory, Blood, Biochemical, Endpoint, B-cell receptor complex, Serum, pigmented epithelium, peptide, Polypeptides, Laboratory Markers, Techniques, peptido, anatomical structure inflammation, Biological, Method, responsivity, GRP1, Grp1, 3, NUP96, Analysis, epithelium, IKKg, KEY, Key, Mass Spectrum Analysis, me75, Divorced, inflammatory response, Man (Taxonomy), peptides, Analyses, membrane bound, PTPSTEP, hypoplasia, pigmented retina, Divorces, D17Mit170, free, T1, DNA cyclobutane dipyrimidine photolyase activity, SUPPRESSOR OF AUXIN RESISTANCE 3, allergic reaction, Immune, Markers, inflammatory disease, Methodological Studies, scientific observation, Viral Markers, l(2)k08110, keratinized cell of epidermis, B-lymphocyte receptor complex, GPH, PRE, Viral, Detection Limits, Surrogate Endpoint, Modern, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, Biochemical Markers, Procedure, Blood Serum, Tl3, antibodies, Biologic Marker, Tl2, Spectrum Analysis, results, Multiple Reaction Monitoring, Spectroscopy, DmIKKgamma, retinal pigment, dIKK, Marker, Kenny, 3.1.3.48, dipyrimidine photolyase (photosensitive), retinal pigment layer, End Points, Step, CG11628, IKK-gamma, Spectrometry, Striatum-enriched protein-tyrosine phosphatase, Immunologic, Methodological, Laboratory Marker, Methodological Study, human, summary, immunoglobulin, phr A photolyase activity, DmelCG16910, B cell receptor accessory molecule complex, DNA-photoreactivating enzyme, Specificity and Sensitivity, Biochemical Marker, Limits of Detection, STEP, B lymphocyte receptor complex, Polypeptide, inflammation of anatomical structure, Innate, Inflammatory Response, malpighian cell, human being, GRP1/cytohesin 1, Procedures, Clinical Markers, Clinical Marker, number, Gene, Spectrum Analyses, photoreactivating enzyme activity, presence, dIKK-gamma, Surrogate End Points, Human, CG11633, cytohesin/GRP1, Surrogate Markers, antibody, method, BCR complex, Homo sapiens, reduced, sensitive, DmIKK-gamma, method used in an experiment, Mass, Studies, Gene Products, stratum pigmentosa retinae, Low, dmIKKgamma, tiny, IKK[[gamma]], Separated, immunoglobulin complex, Technique, Man, sensitivity, Mass Spectroscopy, Biomarker, reactivity, anatomical systems, MOS3, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, Clinical, MRM, PRECOCIOUS, Biological Marker, Blood., F23A5.3, l(2)SH2 0323, inflammation, Study, Immunologic Markers, Neural-specific protein-tyrosine phosphatase, IKK, Innate Inflammatory Response, synopsis, Sensitivity, peptidos, Immunologic Marker, F23A5_3, Biologic, small, measuring, cou, Serum Markers, Proteins, End Point, stepk, backward, Peptide, Immune Marker, polypeptide, IKKgamma, count in organism, MODIFIER OF SNC1, MS, Lr, Cytokine, Surrogate End Point, chemical analysis, Protein, deoxyribonucleic photolyase activity, Detection Limit, l(2)SH0323, Mass Spectrum Analyses, Biologic Markers, opsonin activity, CYH1, Mass Spectrum, Separation, inflammatory disorder, Serum Marker, RPE, Exhibit, underdeveloped, photolyase activity, Dmikkgamma, Surrogate, Separations, Endpoints, Specificity, Keratinocyte, CG16910, Surrogate Marker, DmelCG11628, p. pigmentosa retinae, plan specification, Protein Gene Products, Gene Proteins, Innate Inflammatory Responses, Modern Man, Bra, B cell receptor activity, Peptid, deoxyribonucleate pyrimidine dimer lyase (photosensitive), assay, response, Serums, reversed, Immune Markersopsonin activity, Mass Spectrum Analysis, primary cell culture, Mass Spectrum, Secretomes, human being, Man (Taxonomy), Analyses, membrane bound, Modern, Spectrometry, Protein Secretomes, Primary Cell Cultures, B-cell receptor complex, Primary, Spectrum Analyses, antibodies, Spectrum Analysis, human, Human, Spectroscopy, antibody, immunoglobulin, BCR complex, MS, B cell receptor accessory molecule complex, Cytokine, Homo sapiens, Secretome, Modern Man, B lymphocyte receptor complex, primary cell culture sample, Protein, B cell receptor activity, Mass, Mass., Cell Cultures, Protein Secretome, Analysis, B-lymphocyte receptor complex, Cell Culture, immunoglobulin complex, Man, Mass Spectrum Analyses, Mass Spectroscopyopsonin activity, Mass Spectrum Analysis, primary cell culture, Mass Spectrum, Secretomes, human being, Man (Taxonomy), MRM, Analyses, membrane bound, Modern, Spectrometry, Protein Secretomes, Primary Cell Cultures, B-cell receptor complex, Primary, Spectrum Analyses, antibodies, free, Spectrum Analysis, human, Human, Multiple Reaction Monitoring, Spectroscopy, antibody, immunoglobulin, BCR complex, MS, B cell receptor accessory molecule complex, Cytokine, Homo sapiens, Secretome, Modern Man, B lymphocyte receptor complex, primary cell culture sample, Protein, B cell receptor activity, Mass, Mass., Cell Cultures, Protein Secretome, Analysis, B-lymphocyte receptor complex, Cell Culture, immunoglobulin complex, Man, Mass Spectrum Analyses, Mass SpectroscopyBG:DS00941.4, l(3)LG9, CG7793, determination, Ass-1, Blood, Sry-rDM33, Sry-rDM36, l(2)Sos, Measure, Serum, Cost-Minimization, peptide, Polypeptides, Techniques, peptido, Method, AA408052, Sry-rDM23, fold, Analysis, Cost Comparison, Fs(3)Hor, r3d1, E(sev)2A, Immunoassays, Mass Spectrum Analysis, DmelCG2684, me75, Man (Taxonomy), DM10, peptides, Analyses, E(var)189, Cost-Minimization Analyses, Assays, R3D1-S, L-lysine:oxygen 6-oxidoreductase (deaminating) activity, Comparison, R3D1-L, NTef2, D17Mit170, T1, Conditioned Culture Media, Sry-rDM10, allergic reaction, ASS, Immunochromatographic Assay, Cost Analysis, Methodological Studies, R3D1, scientific observation, keratinized cell of epidermis, Comparisons, fish, Affordabilities, Sox70D, DM33, DM36, Modern, Aptitudes, Procedure, Blood Serum, DmelCG7793, Tl3, Tl2, Spectrum Analysis, Conditioned Medium, Fs(3)Sz11, Multiple Reaction Monitoring, Ability, Spectroscopy, DM23, dme-SOS, median, Medium, finances, br24, Sry-rDM63, dSos, br25, Conditioned Culture Medium, LOQS, 34Ea, Spectrometry, salaries, Immunochromatographic, Methodological, Methodological Study, human, early, Specificity and Sensitivity, Horka, CG2684, Fs(3)Horka, Polypeptide, financial management, Cost Analyses, malpighian cell, SOXB2.1, human being, Procedures, dRax, Cost Comparisons, Spectrum Analyses, EK2-8, Human, Lod, loD, Loqs, method, DM63, Cost Minimization Analysis, Homo sapiens, Culture Medium, sensitive, method used in an experiment, Mass, Studies, CG6866, DmF2, Low, CG5893, Technique, Man, sensitivity, l(2)br25, Mass Spectroscopy, l(2)br24, lod, anatomical systems, MRM, Conditioned Media, Cost Measures, Affordability, loq, l(2)k06321, Blood., Measures, financing, Study, funding, Abilities, LodA, TRBP, Sensitivity, peptidos, l(2)34Ea, SOS, 2.3, fees, measuring, DmelCG5893, Sox B2-1, Immunochromatographic Assays, DmelCG6866, cou, marinocine, Assay, Sox70, sos, Peptide, polypeptide, MS, Lr, Cytokine, Media, SOX70D, l(2)k05224, chemical analysis, Conditioned, Mass Spectrum Analyses, Sos1, SOXDP, Costs and Cost Analyses, Mass Spectrum, Costs, Exhibit, L-lysine-epsilon-oxidase activity, Cost, EY2-3, Specificity, cg6866, Cost-Minimization Analysis, Talents, Pricing, Keratinocyte, Lds, plan specification, cost, SOX70, Su(tor)2-2, Talent, Modern Man, Bra, Peptid, assay, Cost Measure, Serums, CG6866)0falseAntibody-free multiplex measurement of 23 human cytokines in primary cell culture secretome using targeted multiple reaction monitoring mass spectrometryCytokines are key mediators of inflammation that commonly serve as biomarkers to monitor immune responses and inflammatory disease. Cytokines are commonly measured by immunoassays, which have limited multiplexing capacity, are costly and can exhibit cross-reactivity. Although multiple reaction monitoring (MRM) mass spectrometry is a cost-effective method for multiplex measurement of peptides, the ability to detect the relatively low level of secreted cytokines in a complex biological matrix without extensive processing remains to be fully evaluated. We used human keratinocyte conditioned media as an experimental system to evaluate the assay linearity, reproducibility and limit of detection of a MRM assay targeting 23 human cytokines with and without prior 2D chromatographic separation. Without antibody enrichment or immunodepletion of high abundant proteins, the MRM assay could detect 21 cytokines by two or more unique peptides, and two cytokines by a single unique peptide. In a serum-free matrix, the limit of detection and quantification for the 79 monitored cytokine peptides ranged from 7 to 507pg/mL and 24 to 1689 pg/mL, respectively. The presence of serum reduced assay sensitivity however this could be compensated for by high-pH reversed-phase fractionation. Furthermore, the assay shows good replicate consistency with 8% intra- and 12% inter-day coefficient of variations, with increases in assay variability by circa 10% when a pre-fractionation step is included. In summary, our results show potential utility of a multiplex cytokine MRM for routine measurement of secreted cytokines in cellular experiments. Additional enrichment steps will be required in high complexity matrices such as serum.Wed Feb 19 00:00:00 GMT 2020PXD015448960632069036