Fig. 1
From: Heparin-enriched plasma proteome is significantly altered in Alzheimer’s disease
Heparin enrichment of the plasma proteome. A Step-by-step process of heparin enrichment and subsequent mass spectrometry (MS) analysis. Plasma samples were subjected to heparin enrichment, yielding three distinct fractions: DP input (n = 3), Hp-depleted FT (n = 3), and Hp-enriched fraction (n = 3). Each fraction underwent either Coomassie Blue staining, western blotting, or trypsin digestion before subsequent label-free MS analysis utilizing an Orbitrap Eclipse mass spectrometer. B Coomassie Blue staining and western blotting was performed in triplicates for all fractions. A reduction of the amount of albumin (~ 66 kDa) in the Hp-enriched fraction compared to the DP input and FT was illustrated by Coomassie Blue staining. Enrichment was also determined by western blotting for thrombin (~ 75 kDa) and APOE (~ 34 kDa) in the Hp-enriched fraction. C MS base peak chromatograms of each fraction reveal the variation in sample complexity. The Hp-enriched fraction exhibits a notable increase in sample complexity compared to the DP input and Hp-depleted FT fractions, reflecting successful enrichment of low-abundant proteins in the Hp-enriched fraction. D Total number of peptide spectral counts (reported as the maximum percentage) for albumin, thrombin, and APOE in each fraction. Actual spectral counts are labeled within columns. Albumin is significantly depleted from the Hp-enriched fraction compared to the DP input and FT. Conversely, thrombin and APOE are significantly enriched in this fraction. ANOVA with Tukey post-hoc correction was used to determine the p-values (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). E The number of proteins identified in each fraction: DP input (N = 611), Hp-depleted FT (N = 527), and Hp-enriched fraction (N = 771), and the degree of protein overlaps among them. F) GO terms for proteins detected in each fraction, using 841 total proteins measured in at least 2 out 3 replicates within each fraction as background, highlighting the enrichment of proteins associated with the "Heparin binding" molecular function in the Hp-enriched fraction. Z-score > 1.3 (p < 0.05) is significant. WB, western blotting; LC–MS, liquid chromatography-mass spectrometry