Fig. 8
Validation of MS discoveries by ELISA and TOMAHAQ assays a) Validation of MS discoveries by ELISA assay. Dots overlaid onto boxplots showing expression level of the AD biomarker candidate GPNMB in CSF quantified by ELISA assay. Seven healthy control and 7 AD samples were analyzed. The p value of the DE analysis between AD and healthy control by Student’s t-test is displayed on the top of the plots. X-axis shows sample groups, y-axis indicates the ELISA measurement of GPNMB concentration in CSF (ng/ml). Boxplot center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, expression levels of individual samples. b Scatter plot shows the correlation between the ELISA and the discovery MS data. Pearson correlation coefficient (r) is displayed. X-axis shows the Log2TMT ion intensities of GPNMB quantified by MS. Y-axis indicates the CSF concentration of GPNMB (ng/ml) quantified by ELISA. c Workflow for the TOMAHAQ targeted MS validation assay. A synthetic trigger peptide was spiked into the mixture of multiplexed samples to validate the quantification of a candidate biomarker peptide. The synthetic peptide and native peptide were co-eluted, and the synthetic peptide was presented at high concentration, triggering the MS instrument to quantify the native peptide by MS3 using a predefined isolation offset. MS3 ions were produced by pre-determined y or b ions from targeted MS2 spectra, and the resulting reporter ions were applied for the quantification of the targeted biomarker candidate. d Comparison of the TOMAHAQ results and the discovery MS data. e Scatter plot shows the correlation between the TOMAHAQ and the discovery MS results of AK2 in the human CSF samples. Pearson correlation coefficient (r) is displayed. X- and Y- axes indicate the Log2 TMT intensities from the discovery MS and TOMAHAQ assay, respectively. f Scatter plot shows the correlation between the TOMAHAQ and the discovery MS results of PCK2 in the human CSF samples