In Depth Quantification of Extracellular Matrix Proteins from Human Pancreas

Abstract

Extracellular matrix (ECM) is an important component of the pancreatic microenvironment which regulates β cell proliferation, differentiation, and insulin secretion. Protocols have recently been developed for the decellularization of the human pancreas to generate functional scaffolds and hydrogels. In this work, we characterized human pancreatic ECM composition before and after decellularization using isobaric dimethylated leucine (DiLeu) labeling for relative quantification of ECM proteins. A novel correction factor was employed in the study to eliminate the bias introduced during sample preparation. In comparison to the commonly employed sample preparation methods (urea and FASP) for proteomic analysis, a recently developed surfactant and chaotropic agent assisted sequential extraction/on pellet digestion (SCAD) protocol has provided an improved strategy for ECM protein extraction of human pancreatic ECM matrix. The quantitative proteomic results revealed the preservation of matrisome proteins while most of the cellular proteins were removed. This method was compared with a well-established label-free quantification (LFQ) approach which rendered similar expressions of different categories of proteins (collagens, ECM glycoproteins, proteoglycans, etc.). The distinct expression of ECM proteins was quantified comparing adult and fetal pancreas ECM, shedding light on the correlation between matrix composition and postnatal β cell maturation. Despite the distinct profiles of different subcategories in the native pancreas, the distribution of matrisome proteins exhibited similar trends after the decellularization process. Our method generated a large data set of matrisome proteins from a single tissue type. These results provide valuable insight into the possibilities of constructing a bioengineered pancreas. It may also facilitate better understanding of the potential roles that matrisome proteins play in postnatal β cell maturation.

Keywords: FASP; SCAD; decellularized pancreas; dimethylated leucine (DiLeu); extracellular matrix (ECM); isobaric quantitation; label-free quantitation (LFQ); matrisome; urea.

Figure 1.

Comparison of sequence coverage achieved (A) and Venn diagram showing overlap of matrisome proteins identified among different sample preparation strategies (B).

Figure 2.

Percentage of relative intensity quantified by DiLeu labeling for nonmatrisome proteins and each matrisome subcategory from adult pancreas before and after decellularization process (A) and gene ontology enrichment analysis of quantified proteins, top 35 annotations were listed (B).

Figure 3.

Venn diagram showing overlap of matrisome proteins identified in fetal pancreas between label-free method and DiLeu labeling (A) and number of total proteins and matrisome-related proteins identified in fetal pancreas (B).

Figure 4.

Number of total proteins (A) and matrisome-related proteins (B) identified in fetal and adult pancreas before and after decellularization process using LFQ method.

Figure 5.

Percentage of LFQ intensity for each matrisome subcategory in native pancreas (A) and decellularized pancreas (B).

Figure 6.

Heatmap visualization of protein expression profiles of human pancreas from various ages before and after decellularization process. A dendrogram of two pancreas ages (fetal and adult) with two experimental conditions (native (U) and decellularized (F) pancreas) (17U, 21U, and 24U for adult native; 4f-U, f5-U, and f6-U for fetal native; 17F, 21F, and 24F for adult decell; f4, f5, and f6 for fetal decell) was shown at the top. Protein expression values were Z-score normalized prior to clustering. Hierarchical clustering of protein expression profiles identified four groups. The cluster of proteins with higher abundance in fetal pancreas was highlighted in pink. The cluster of proteins with higher abundance in adult tissues was highlighted in yellow.