Proteomic Analysis of Human Serum Proteins Adsorbed onto Collagen Barrier Membranes

Abstract

Collagen barrier membranes are frequently used in guided tissue and bone regeneration. The aim of this study was to analyze the signature of human serum proteins adsorbed onto collagen membranes using a novel protein extraction method combined with mass spectrometry. Native porcine-derived collagen membranes (Geistlich Bio-Gide^®, Wolhusen, Switzerland) were exposed to pooled human serum in vitro and, after thorough washing, subjected to protein extraction either in conjunction with protein enrichment or via a conventional surfactant-based method. The extracted proteins were analyzed via liquid chromatography with tandem mass spectrometry. Bioinformatic analysis of global profiling, gene ontology, and functional enrichment of the identified proteins was performed. Overall, a total of 326 adsorbed serum proteins were identified. The enrichment and conventional methods yielded similar numbers of total (315 vs. 309), exclusive (17 vs. 11), and major bone-related proteins (18 vs. 14). Most of the adsorbed proteins (n = 298) were common to both extraction groups and included several growth factors, extracellular matrix (ECM) proteins, cell adhesion molecules, and angiogenesis mediators involved in bone regeneration. Functional analyses revealed significant enrichment of ECM, exosomes, immune response, and cell growth components. Key proteins [transforming growth factor-beta 1 (TGFβ1), insulin-like growth factor binding proteins (IGFBP-5, -6, -7)] were exclusively detected with the enrichment-based method. In summary, native collagen membranes exhibited a high protein adsorption capacity in vitro. While both extraction methods were effective, the enrichment-based method showed distinct advantages in detecting specific bone-related proteins. Therefore, the use of multiple extraction methods is advisable in studies investigating protein adsorption on biomaterials.

Keywords: collagen membranes; guided bone regeneration; guided tissue regeneration; mass spectrometry; protein extraction.

Figure 1

Comparison between the two protein extraction methods. (A) Venn diagram illustrating the numbers of common and exclusive proteins between the ENRICH (EN) and RapiGest (RG) methods. (B) Volcano plot showing differentially expressed proteins (DEPs) (adjusted p-value) vs. magnitude of expression change (log2-fold change) between RG (n = 131 DEPs) and EN (n = 70 DEPs).

Figure 2

Hierarchical clustering analysis of adsorbed serum proteins in the ENRICH (EN) and RapiGest groups (RG). (A) All adsorbed proteins. (B) Differentially expressed proteins. (C) Bone-related proteins. Intensity values (protein abundances) are represented as colors ranging from low (green) to high (red); n = 3 technical replicates in each group.

Figure 3

GO analysis of serum proteins adsorbed on collagen membranes. (A) Results of enrichment analysis presented in the form of a Manhattan plot, where the X-axis shows the functional terms grouped by the color code of the source database used, and the Y-axis shows the enrichment adjusted p-values in a negative decimal logarithm scale. Dots in the graph indicate all enriched terms meeting the significance criterion of p < 0.05, while highlighted dots represent terms filtered by the criterion of top 10 terms. (B) The graphs show detailed results of the enriched terms highlighted in the Manhattan plot along with the statistical significance [−log10(p-value)] and number of common proteins (counts) belonging to the enriched term, according to molecular function (MF), biological process (BP), and cellular component (CC) categories.

Figure 4

Functional enrichment analysis of adsorbed serum proteins. Graphs showing the percentage representation and significance levels (p-values) of the top 10 enriched groups under “Biological processes”, “Molecular function”, and “Cellular components” categories. p < 0.05 indicates statistically significant enrichment.