Targeting Interferon Alpha Subtypes in Serum: A Comparison of Analytical Approaches to the Detection and Quantitation of Proteins in Complex Biological Matrices

The targeted detection and quantitation of proteins in complex biological fluids such as blood is as analytically challenging as it is crucial for biomedical research. Antibody-based techniques such as the ELISA are the current standards for such measurements, having in favorable cases high specificity and pg/mL detection limits. Long development timelines and susceptibility to cross reactivity have led researchers to investigate mass spectrometric alternatives. The literature contains diverse schemes for sample preparation and multiple platforms for mass spectrometric detection. Critical evaluations of competing technologies are, however, badly needed. Taking closely related subtypes of the pro-inflammatory cytokine interferon alpha as a test case, we compared a sample preparation workflow based on affinity enrichment to one based on generic multidimensional chromatography, and evaluated mass spectrometric techniques using tandem mass spectrometry on low resolution ion traps, high resolution “accurate mass tags,” and triple quadrupole selective reaction monitoring. Each workflow and detection method proved capable of detecting and discriminating between these proteins at or below the ng/mL level in human serum. Quantitation by isotope dilution was evaluated using full length protein as the internal standard. Both triple quadrupole selected reaction monitoring and orbitrap selected ion monitoring produced linear calibration curves from 1 ng/mL to 1 μg/mL, with lower limits of quantitation below 5 and 50 ng/mL, respectively

Targeting Interferon Alpha Subtypes in Serum: A Comparison of Analytical Approaches to the Detection and Quantitation of Proteins in Complex Biological Matrices

The targeted detection and quantitation of proteins in complex biological fluids such as blood is as analytically challenging as it is crucial for biomedical research. Antibody-based techniques such as the ELISA are the current standards for such measurements, having in favorable cases high specificity and pg/mL detection limits. Long development timelines and susceptibility to cross reactivity have led researchers to investigate mass spectrometric alternatives. The literature contains diverse schemes for sample preparation and multiple platforms for mass spectrometric detection. Critical evaluations of competing technologies are, however, badly needed. Taking closely related subtypes of the pro-inflammatory cytokine interferon alpha as a test case, we compared a sample preparation workflow based on affinity enrichment to one based on generic multidimensional chromatography, and evaluated mass spectrometric techniques using tandem mass spectrometry on low resolution ion traps, high resolution “accurate mass tags,” and triple quadrupole selective reaction monitoring. Each workflow and detection method proved capable of detecting and discriminating between these proteins at or below the ng/mL level in human serum. Quantitation by isotope dilution was evaluated using full length protein as the internal standard. Both triple quadrupole selected reaction monitoring and orbitrap selected ion monitoring produced linear calibration curves from 1 ng/mL to 1 μg/mL, with lower limits of quantitation below 5 and 50 ng/mL, respectively

Figure S2 from A KDM5 Inhibitor Increases Global H3K4 Trimethylation Occupancy and Enhances the Biological Efficacy of 5-Aza-2′-Deoxycytidine

DAC & KDM5i induced expression changes</p

Figure S1 from A KDM5 Inhibitor Increases Global H3K4 Trimethylation Occupancy and Enhances the Biological Efficacy of 5-Aza-2′-Deoxycytidine

KDM5i increases H3K4 trimethylation alone and in combination with DAC.</p

Supplementary Tables from A KDM5 Inhibitor Increases Global H3K4 Trimethylation Occupancy and Enhances the Biological Efficacy of 5-Aza-2′-Deoxycytidine

Contains Tables S1 and S2. Supplementary Table 1: List of PCR primers. Supplementary Table 2: Sensitivity of Breast Cancer Cell Lines to CPI-455 Cell Viability was measured following exposure to CPI-455 for 8 days.</p

Figure S3 from A KDM5 Inhibitor Increases Global H3K4 Trimethylation Occupancy and Enhances the Biological Efficacy of 5-Aza-2′-Deoxycytidine

KDM5i alone and in combination with DAC upregulates cancer hallmark & immunomodulatory pathways</p

Figure S5 from A KDM5 Inhibitor Increases Global H3K4 Trimethylation Occupancy and Enhances the Biological Efficacy of 5-Aza-2′-Deoxycytidine

Effects of KDM5i and DAC on enhancers and de novo super-enhancers</p

Supplementary Information from A KDM5 Inhibitor Increases Global H3K4 Trimethylation Occupancy and Enhances the Biological Efficacy of 5-Aza-2′-Deoxycytidine

Legends for Supplementary Figures and Tables</p

Figure S4 from A KDM5 Inhibitor Increases Global H3K4 Trimethylation Occupancy and Enhances the Biological Efficacy of 5-Aza-2′-Deoxycytidine

Differential H3K4me3 enrichment profiling after KDM5i, DAC, or both</p

Figure S6 from A KDM5 Inhibitor Increases Global H3K4 Trimethylation Occupancy and Enhances the Biological Efficacy of 5-Aza-2′-Deoxycytidine

KDM5i and DAC do not synergize in MCF10A, nor alter cell cycle in MCF-7</p