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. 2016 Feb;15(2):726-39.
doi: 10.1074/mcp.O115.054940. Epub 2015 Nov 30.

Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling

Jacob J Kennedy  1 , Ping Yan  1 , Lei Zhao  1 , Richard G Ivey  1 , Uliana J Voytovich  1 , Heather D Moore  1 , Chenwei Lin  1 , Era L Pogosova-Agadjanyan  1 , Derek L Stirewalt  1 , Kerryn W Reding  2 , Jeffrey R Whiteaker  1 , Amanda G Paulovich  3
Affiliations

Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling

Jacob J Kennedy et al. Mol Cell Proteomics. 2016 Feb.

Abstract

A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks.

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Figures

Fig. 1.
Fig. 1.
Source of targets for IMAC-MRM assay development. Phopshopeptides were selected for IMAC-MRM assay development from three sources: (1) Single step IMAC enrichment of cell lysates coupled to LC-MS/MS, (2) offline basic reverse-phase fractionation prior to IMAC enrichment and LC-MS/MS, (3) survey of published or publically available LC-MS/MS datasets of phosphorylation. For each source the types of lysates used are shown along with the filter criteria applied.
Fig. 2.
Fig. 2.
Method optimization for robust IMAC enrichment of phosphopeptides using magnetic beads. IMAC sample handling was refined by using magnetic beads to produce a robust, reproducible procedure for phosphopeptide enrichment. A, Comparison of peak area ratio and absolute peak area for phosphopeptides enriched using agarose IMAC beads and magnetic agarose IMAC beads. Enrichment was performed from 500 μg lysate for agarose and 200 μg lysate for magnetic beads. Spike levels of heavy stable isotope-labeled peptides were adjusted according to input material, so 2.5 times more heavy peptides were added to the agarose bead samples. Peak areas for heavy peptides are plotted in orange, light peptides are plotted in blue. B, Magnetic beads were regenerated by collecting used beads, and recoupling the Fe metal. Beads were re-used to enrich phosphopeptides from two identical aliquots of 200 μg cell lysate. C, Magnetic beads from different manufacturer lots were compared by enriching phosphopeptides from the aliquots of the same lysate. Error bars are the standard deviation of process triplicates. Regression for peak areas was performed using a y-weighted linear fit. The correlation coefficient (R2), slope (m), and number of phosphopeptides measured (n) are reported on each plot.
Fig. 3.
Fig. 3.
IMAC-MRM assay validation. A, Representative response curve for the phosphopeptide NYPpSQEELIK (pS1524 on BRCA1). Each point measures the peak area ratio of heavy to light peptide (heavy spiked into 200 μg aliquots of cell lysate digest; light measured as endogenous analyte). The inset shows the response curve plotted on logarithmic scale. Error bars are the standard deviation of three process replicates. B, Distribution of the LLOQ for 107 validated assays. Limits of quantification were determined by the lowest point measured with CV less than 20%. C, Assays were validated by measuring endogenous phosphopeptide in five replicates on each of 5 days. Intra-assay variation reports the distribution of average %CV of five within-day process replicates. Inter-assay variation is the distribution of average %CV for between-day measurements. Total variation is the distribution of the square root of the sum of squares for intra- and inter-assay variation. Data for 107 validated assays are plotted. Box plots show the median (bar), inner quartiles (box), 5–95th percentiles (line), and outliers (whisker).
Fig. 4.
Fig. 4.
Phosho-pharmacodynamic responses of human PBMCs responding to genotoxic stress. The multiplexed IMAC-MRM assay was used to quantify phospho-signaling in PBMC treated with 10 Gy IR (mock-treated samples were harvested at 1 h). Blood from a single donor was obtained at three different points in time, separated by several weeks. PBMCs (±IR) from the first blood draw were analyzed by IMAC-MRM in complete process triplicate, and the kinetic response curves for representative analytes (ATMpS2996, ATMpS367, NBS1pS343, BRCA1pS1524, SMC3pS1083, and TP53pS315) are displayed. Black line shows results from the first draw (analyzed in complete process triplicate). The error bars represent the standard deviation of the three process replicates. The green line shows results from the second draw. The orange line shows results from the third draw. The dotted line represents the LLOQ. Points detected below LLOQ are plotted with orange error bars for delineation.
Fig. 5.
Fig. 5.
Activity of the DDR measured by the multiplexed IMAC-MRM assay. Response following DNA damage is plotted as a heat map for endogenous-detected analytes in the multiplex IMAC-MRM panel (using R script heatmap.2). Values are the log2 median phosphopeptide concentration of three biological replicates relative to the concentration measured in the mock-treatment. For visualization in the heat maps, data points from endogenous levels below the LLOQ were imputed using the LLOQ peak area for the endogenous signal.
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