Fast and Simple Protocols for Mass Spectrometry-Based Proteomics of Small Fresh Frozen Uterine Tissue Sections

Abstract

Human tissues are an important link between organ-specific spatial molecular information, patient pathology, and patient treatment options. However, patient tissues are uniquely obtained by time and location, and limited in their availability and size. Currently, little knowledge exists about appropriate and simplified protocols for routine MS-based analysis of the various types and sizes of tissues. Following standard procedures used in pathology, we selected small fresh frozen uterine tissue samples to investigate how the tissue preparation protocol affected the subsequent proteomics analysis. First, we observed that protein extraction with 0.1% SDS followed by extraction with a 30% ACN/urea resulted in a decrease in the number of identified proteins, when compared to extraction with 30% ACN/urea only. The decrease in the number of proteins was approximately 55% and 20%, for 10 and 16 μm thick tissue, respectively. Interestingly, the relative abundance of the proteins shared between the two methods was higher when SDS/ACN/urea was used, compared to the 30% ACN/urea extraction, indicating the role of SDS to be beneficial for protein solubility. Second, the influence of tissue thickness was investigated by comparing the results obtained for 10, 16, and 20 μm thick (1 mm²) tissue using urea/30% ACN. We observed an increase in the number of identified proteins and corresponding quantity with an increase in the tissue thickness. Finally, by analyzing very small amounts of tissues (∼0.2 mm²) of 10, 16, and 20 μm thickness, we observed that the increase in tissue thickness resulted in a higher number of protein identifications and corresponding quantitative values.

Figure 1

Schematic overview of the current study. (a) 10, 16, and 20 μm thick FF human uterus tissue were used in the study. (b) Proteins from the tissues were extracted using 3 different solutions varied by detergent (SDS) and organic solvent (ACN). (c) Protein digestion was performed over 18 h using a single portion of trypsin (Protocols 1, 2, and 4) or for an additional 4 h after the addition of second portion of trypsin (Protocols 3 and 5).

Figure 2

Summary of the results showing qualitative and quantitative comparison of five protocols for protein retrieval from 10 μm thick FF human uterus tissue. Venn diagrams show the distribution of identified proteins in biological replicates using Protocols 1 to 5, and correlation of the NSAF values between replicates is shown using Pearson r coefficient.

Figure 3

Overview of protein identifications and their quantitative values using various protocols and tissue amounts. (a) Relative comparison of identified proteins from 10 μm thick tissue after extraction and digestion using Protocols 1 to 5. Addition of SDS into the protein extraction buffer revealed a decrease in the number of identified proteins (Protocol 1 as compared to Protocols 2–5). Similar results were obtained for 16 μm thick tissue (Figure S-4, Supporting Information). (b) Quantitative comparison of shared-protein abundance by NSAF values after extraction and digestion using Protocol 2 for quantitation in the 10, 16, and 20 μm thick tissue. Abundance of the proteins after tissue processing using Protocol 1 and Protocol 2 are shown for: (c) 10 μm and (d) 16 μm thick FF human uterus tissue. Results are shown as mean ± SD.

Figure 4

Quantitative comparison of protein abundance by NSAF values for 0.2, 0.6, 0.8, 1.2, 1.6, and 2.0 mm² of 10, 16, and 20 μm thick FF uterus tissue. Results are obtained after extraction and digestion of the proteins using Protocol 2. (a) Number of identified proteins; (b) NSAF values compared across different samples.

Figure 5

Distribution of the proteins identified from 3 biological replicates in 10 μm thick FF human uterus tissue after extraction and digestion using Protocols 1 and 2. (a) Sequence coverage; (b) NSAF values; and (c) GRAVY index. Results are shown as mean ± SD.