Fig. 2: SEC-MX enables phosphopeptide enrichment.

A Overview of the experimental workflow of SEC-MX. The cells were lysed, fractionated by size-exclusion chromatography, digested, and labeled. After pooling, 80% of each 18-fractions mix were allocated for phosphopeptide enrichment and measurement of phSEC, while the remaining 20% were processed for gSEC. Created in BioRender. Jovanovic, M. (2024). [https://BioRender.com/ s07v514]⁷⁷. B Venn diagram showing the overlap of protein identifications between the gSEC and phSEC datasets. C Distribution of Pearson correlation coefficients between gSEC and phSEC elution profiles for each overlapping peptide in the HEK293 dataset, per replicate (replicate 1 in blue, replicate 2 in orange, as indicated in the key). 456 / 627 and 343 / 522 peptides have correlation coefficients greater than 0.5, in replicate 1, and 2, respectively. D Heatmap representation of the elution profiles of peptides overlapping between gSEC and phSEC (HEK293 data). The peptide number (n = 909) represents the number of rows and is based on the intersection of peptide identifications across the datasets (gSEC/phSEC), in the HEK293 signal averaged between replicates. Columns represent fractions. Signal is scaled so that the max elution peak per protein is represented in red. Rows in both heatmaps are arranged in the same order. E Elution traces for CCT complex members identified in gSEC and phSEC from the average of two replicates in HEK293 cells. Traces are color-coded by subunit, as indicated to the right of the plot. (Similar results were obtained for HCT116 cells, shown in Supplementary Fig. 3E).