MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/x01/MSV000079983/primary200OK0000ProteomicsDr Christopher GernerQ ExactiveHomo Sapiens (ncbitaxon:9606)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=a130255c964641d6b0b37b918f56ae7bMassIVE26UNIMOD:4 - "Iodoacetamide derivative."UNIMOD:1 - "Acetylation."UNIMOD:Determination of secreted proteins provides highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low abundant molecular species still rely on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges as well. In the current study, we have identified several cytokines and chemokines which were induced in primary human umbilical vein endothelial cells upon inflammatory activation. Based on the high-resolution mass spectrometry data obtained with a Q Exactive orbitrap, we built an MRM method to quantify the most relevant molecules selected from the screening experiment. All experimental data are available via ProteomeXchange, PXD002211/12, and Panorama ( www.panoramaweb.org ). Using nano-flow Chip-HPLC coupled to a 6490 triple-quadrupole MS for MRM analyses, we achieved calibration curves covering a linear range of four orders of magnitude and detection limits in the low attomol per microliter concentration range. Carryover was consistently less than 0.005 %, the accuracy was between 80 and 120 %, and the median coefficient of variation for LC/MS was only 2.2 %. When including the variance of quantification introduced by cell culture and digestion, the coefficient of variation was less than 20 % for most peptides. With appropriate marker molecules, we monitored typical variations introduced by cell culture caused by differences in cell numbers, proliferative states, and cell death. As a result, here, we present a robust and efficient MRM-based assay for the accurate and sensitive determination of cytokines and chemokines representative for functional cell states and including comprehensive quality controls. Graphical Abstract Work flow diagram: Data processing steps beginning with orbitrap-based shotgun data acquisition and MaxQuant data analysis, followed by peptide and transition selection for MRM analysis using Skyline and experimental validation using triple quadrupole MS.Quantification of cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters.Muqaku Besnik B, Slany Astrid A, Bileck Andrea A, Kreutz Dominique D, Gerner Christopher CHuman, Multiple Reaction Monitoring, MRM., Cytokine, human being, Man (Taxonomy), Homo sapiens, Modern Man, Modern, Man, human, humans, CellEndothelial Cells, determination, selection process, Dm NinaC, NINA C, CG5125, Model Calibration, dmTAF[[II]]230, peptide, Polypeptides, Techniques, peptido, Method, High Performance Liquid Chromatography, Chemokine, Analysis, Gpi-1, Chemotactic Cytokine, 2310040B03Rik, Mass Spectrum Analysis, me75, TFIID TAF250, peptides, Analyses, cel, Nlk, Chip, ChIP, CHIP, procedures, µl, D17Mit170, T1, HPLC, allergic reaction, chip, reaction, Nina C, High-Performance, Methodological Studies, scientific observation, n, Chromatography, AW046544, High-Performance Liquid, High Speed Liquid, necrosis, CT16120, NK/GPI, Gpi, dTAF[[II]]230, l(2)k04405, Liquid Chromatography, TAF200, ul, TAFII-250, Procedure, TAF250/230, Tl3, antibodies, Tl2, Spectrum Analysis, Multiple Reaction Monitoring, DmelCG5125, Spectroscopy, TAFII250, mOC-X, Intercrines, Intercrine, median, NK|GPI, Human Umbilical Vein, activation, SDCCAG7, Sensor, DRONINAC, Culture Technique, Org, ORG, dLdb, Spectrometry, Gpi1-r, Gpi1-s, Methodological, CG17603, Gpi1-t, TAF[[II]], Methodological Study, experimental procedures, data analysis, Human Umbilical Vein Endothelial Cell, Taf250, Specificity and Sensitivity, SR3-5, Sensor Calibration, Cells, Liquid, Sensor Calibrations, Cell Cultures, Polypeptide, experimental procedures., Bglap-rs1, TAF230, Gpi1s, HSPABP2, d230, Chemotactic, HUVEC Cells, Procedures, experimental, AI461847, Chemotactic Cytokines, 2210017D18Rik, dCHIP, Gene, dTAFII250, SCAR16, Spectrum Analyses, EfW1, method, DmelCG5203, dmTAF1, Taf230, sensitive, method used in an experiment, Gene Products, Studies, Mass, Pgi, Low, NINAC, Cell Culture, Technique, sensitivity, Mass Spectroscopy, CG54125, TAF250, study, Taf200, methods, dTAF[[II]]250, NY-CO-7, MRM, NinaC, ninac, cell, Gpi-1r, experimental section, Gpi-1s, Phi, Gpi-1t, Taf1p, HUVEC, High-Performance Liquid Chromatographies, CG 5125, Cell Culture Technique, Study, dTAF250, Abstract, data processing, DmelCG3924, accidental cell death, Ultra Performance Liquid Chromatography, Sensitivity, peptidos, species, UPLC, UBOX1, 2.2, TAF, 0610033N24Rik, Model, PP1131, High-Performance Liquid Chromatography, measuring, TAF[[II]]250, Culture Techniques, cou, Proteins, CG5203, l(3)84Ab, BG:DS00004.13, MF, l(2)04405, Amf, Cell, Peptide, dTAF230, epsilon-COP, polypeptide, Cytokines, Lr, MS, Experiment, Cytokine, p230, Model Calibrations, Protein, chemical analysis, TAF[[II]]250/230, CT42491, TFIID, techniques, Death, Mass Spectrum Analyses, NK, Taf[[II]]250, Mass Spectrum, 10^[-9], TAF[[II]]230, Ldb, LDB, Specificity, Chromatin Immunoprecipitation, TAF[II]250, CG3924, Protein Gene Products, plan specification, Gene Proteins, High Pressure, DmelCG17603, dLDB/Chip, concentration, Data, Calibration, Calibrations, Bra, HUVEC Cell, Peptid, High Performance Liquid, assay, Data Analyses, methodology, TAF1Human, Multiple Reaction Monitoring, MRM., Cytokine, human being, Man (Taxonomy), Homo sapiens, Modern Man, Modern, Man, human, humans, CellEndothelial Cells, determination, selection process, Dm NinaC, NINA C, CG5125, Model Calibration, dmTAF[[II]]230, Polypeptides, Techniques, peptido, Method, High Performance Liquid Chromatography, Chemokine, Analysis, Cell Numbers, Chemotactic Cytokine, 2310040B03Rik, Mass Spectrum Analysis, me75, TFIID TAF250, peptides, Analyses, Densities, cel, Chip, ChIP, CHIP, procedures, µl, D17Mit170, T1, SURGICAL AND MEDICAL PROCEDURES, HPLC, allergic reaction, chip, Nina C, High-Performance, Methodological Studies, scientific observation, n, Chromatography, AW046544, High-Performance Liquid, High Speed Liquid, necrosis, CT16120, dTAF[[II]]230, l(2)k04405, frequency, Liquid Chromatography, TAF200, ul, TAFII-250, Procedure, TAF250/230, Tl3, antibodies, Tl2, Spectrum Analysis, Multiple Reaction Monitoring, DmelCG5125, Spectroscopy, TAFII250, Intercrines, Intercrine, median, activation, Human Umbilical Vein, SDCCAG7, Sensor, DRONINAC, Culture Technique, dLdb, Cell Densities, Spectrometry, Number, Methodological, CG17603, TAF[[II]], Methodological Study, surveillance, morbidity, Human Umbilical Vein Endothelial Cell, Taf250, Specificity and Sensitivity, SR3-5, Sensor Calibration, Cells, Cell Number, Liquid, Sensor Calibrations, Cell Cultures, Polypeptide, TAF230, HSPABP2, d230, Chemotactic, HUVEC Cells, Procedures, Count, Density, Chemotactic Cytokines, 2210017D18Rik, dCHIP, Gene, dTAFII250, SCAR16, Spectrum Analyses, EfW1, Intervention or Procedure, method, DmelCG5203, dmTAF1, Taf230, sensitive, method used in an experiment, Gene Products, Studies, Mass, Low, NINAC, Cell Culture, Technique, sensitivity, Mass Spectroscopy, CG54125, TAF250, study, Taf200, dTAF[[II]]250, NY-CO-7, MRM, NinaC, ninac, interventionDescription, occurrence, cell, prevalence, Interventional, Cell Density, Taf1p, HUVEC, High-Performance Liquid Chromatographies, CG 5125, Cell Culture Technique, Study, dTAF250, DmelCG3924, accidental cell death, Ultra Performance Liquid Chromatography, Sensitivity, peptidos, species, UPLC, UBOX1, 2.2, TAF, 0610033N24Rik, Model, PP1131, incidence, High-Performance Liquid Chromatography, Intervention Strategies, measuring, TAF[[II]]250, Culture Techniques, cou, Proteins, CG5203, l(3)84Ab, BG:DS00004.13, l(2)04405, Cell, Peptide, dTAF230, epsilon-COP, Cytokines, Lr, MS, Experiment, Cytokine, p230, Model Calibrations, Protein, chemical analysis, TAF[[II]]250/230, CT42491, TFIID, techniques, Death, outbreaks, Mass Spectrum Analyses, Intervention, Taf[[II]]250, Mass Spectrum, Counts, 10^[-9], TAF[[II]]230, Ldb, LDB, Numbers, Specificity, Chromatin Immunoprecipitation, TAF[II]250, CG3924, endemics, Protein Gene Products, plan specification, Gene Proteins, Cell Counts, High Pressure, DmelCG17603, dLDB/Chip, TAF1., concentration, Calibration, Calibrations, Bra, HUVEC Cell, Peptid, epidemics, High Performance Liquid, assay, methodology, TAF10PXD002212trueQuantification of cytokines secreted by primary human cells using MRM: evaluation of analytical parameters â?? shotgun secretome data of untreated HUVECsThe determination of secreted proteins may provide highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low-abundant molecular species still relies on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges. In the current study we have identified several cytokines and chemokines which were induced by inflammatory activation of primary human umbilical vein endothelial cells. Based on the high-resolution mass spectrometry data obtained with a Q Exactive orbitrap, we built an MRM method to quantify the most relevant molecules selected from the screening experiment. Using nano-flow Chip-HPLC coupled to a 6490 triple-quadrupole MS for MRM analyses we achieved calibration curves covering a linear range of four orders of magnitude and detection limits in the low attomol per microliter concentration range. Carryover was consistently less than 0.005%, the accuracy between 80% and 120% and the median coefficient of variation for LC/MS was only 2.2%. When including the variance introduced by biological replicates and the digestion procedure, the coefficient of variation was less than 20% for most peptides. Selection of appropriate marker molecules allowed us to monitor typical cell culture variations such as different cell densities, proliferative states and the occurrence of cell death. As a result, here we present a robust and efficient MRM-based assay for the accurate and sensitive determination of cytokines and chemokines representative for functional cell states and including comprehensive quality controls.Fri Jul 22 15:52:00 BST 2016MSV00007998326092402