Fig. 1
Experimental and analysis workflow. (a) 5 CSF samples from different individuals were used for each sample group (MCI and control) as inputs for the proteome profiling and affinity-enrichment (AE)-MS. (b) 5 different conditions were used for the AE-MS approach: (1) control with beads-only; (2) 3S-HS1; (3) 3S-HS 2; (4) HS 1; (5) HS 2. (c) LFQ proteomics was used to profile the proteomes of the 10 CSF samples. The LC-MS/MS data were analyzed by MaxQuant and, from the 759 proteins identified (ID), only those with a minimum of 3 LFQ valid values in at least one group were withheld, resulting in 434 reliably quantified (QT) proteins. Of these proteins, 74 proteins were found only in the MCI samples, 5 only in the control samples, and 355 were shared between both types of samples. When comparing the proteomes, no proteins were found significantly regulated in either the MCI or the control sample groups. (d) AE-MS was used to profile the interactomes of the synthetic HS baits in MCI and control samples. To identify non-specific binders, a beads-only control condition was used. The LC-MS/MS data were identified by MaxQuant, which resulted in the identification of 1,051 proteins. A further selection of proteins with a minimum of 3 valid LFQ values in at least one condition resulted in 698 reliably quantified proteins. To profile the interactomes, proteins with equal or higher levels of expression in the beads-only condition were excluded. Next, proteins upregulated in the 3S-HS 1 or 3S-HS 2 conditions compared to HS 1 and HS 2 conditions were selected as interactors showing preferential binding to HS containing 3-O sulfation (3S-HS interactors). Finally, 3S-HS interactors were compared between the MCI and Control sample groups, but no proteins were found at significantly different levels.
