Figure 3. Venn diagrams depicting overlap (i.e., redundancy) of identified proteins between separate fractions.

(A) Method 1, (B) Method 2, (C) Method 3, (D) Method 4, (E) Method 5, (F) Method 6, (G) demineralization fractions of Method 7, and (H) a hypothetical method that combines three fractions to maximize recovery. In methods 1–5 (A–E), there was a large amount of overlap between the demineralization and solubilization fractions, and most of the unique proteins were found in the demineralization fractions,rendering the solubilization fractions largely redundant. In Method 6 (F), there were slightly more unique proteins in the two solubilization fractions (combined) than in the one demineralization fraction. In Method 7, there was substantial redundancy in the two sequential demineralization incubations, indicating that additional demineralization did not recover substantially new portions of the proteome. Combining three fractions (i.e., 20-HCl-P, 20-N/EDTA-D, and 20-H/SDS-P) into a hypothetical extraction (H) accounted for 51 of the 55 proteins identified across all fractions in this study, with the largest contribution of unique proteins coming from HCl.