Thermal Denaturation of Fresh Frozen Tissue Enhances Mass Spectrometry Detection of Peptides

Abstract

Thermal denaturation (TD), known as antigen retrieval, heats tissue samples in a buffered solution to expose protein epitopes. Thermal denaturation of formalin-fixed paraffin-embedded samples enhances on-tissue tryptic digestion, increasing peptide detection using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS). We investigated the tissue-dependent effects of TD on peptide MALDI IMS and liquid chromatography-tandem mass spectrometry signal in unfixed, frozen human colon, ovary, and pancreas tissue. In a triplicate experiment using time-of-flight, orbitrap, and Fourier-transform ion cyclotron resonance mass spectrometry platforms, we found that TD had a tissue-dependent effect on peptide signal, resulting in a (22.5%) improvement in peptide detection from the colon, a (73.3%) improvement in ovary tissue, and a (96.6%) improvement in pancreas tissue. Biochemical analysis of identified peptides shows that TD facilitates identification of hydrophobic peptides.

Figure 1

Experimental workflow. Fresh frozen tissue sections are subjected to washing steps and optional thermal denaturation to improve digestion efficiency. On-tissue trypsin digestion produces tryptic peptides. Samples can then be coated with a matrix to assist with the extraction and ionization of peptides, followed by matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS). In tandem, microextraction can extract surface peptides for analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Figure 2

Summary and differential abundance analysis of LC-MS/MS data from microextractions. A. The number of unique proteins identified in colon, ovary, and pancreas samples without thermal denaturation (blue) and with thermal denaturation (purple). B. The number of unique peptides identified in colon, ovary, and pancreas samples without thermal denaturation (blue) and with thermal denaturation (purple). C. The Global Average of Hydropathy (GRAVY) value for identified peptides from nondenatured (blue) and thermally denatured (purple) samples in the colon, ovary, and pancreas in which a higher GRAVY score indicates higher average hydrophobicity. Student’s T-Test result **** P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Differential abundance analysis of LC-MS/MS data using label-free quantitation comparing nondenatured and thermally denatured (TD) colon (D,E,F) and ovary (G,H,I). Pancreas tissue was omitted from this analysis because nondenatured pancreas tissue had insufficient protein identifications for statistical comparison. D. Volcano plot visualizing proteins more abundant (in purple) and less abundant (in blue) in TD colon. Proteins with increased abundance in TD tissue included Collagens alpha-1(I), alpha-2(I), alpha-1(III), alpha-1(IV), and alpha-2(IV) (CO1A1, CO1A2, CO3A1, CO4A1, CO4A2), Plectin, Myosin-9 (MYH9), Filamin-A (FLNA), Supervillin (SVIL), and Fibrillin-1 (FBN1). Proteins with decreased abundance in the TD colon include SH3 domain-binding glutamic acid-rich-like protein (SH3L1), Rho GDP-dissociation inhibitor 1 (GDIR1), Heat shock protein beta-1 (HSPB1), 60S ribosomal protein L7a (RL7A), Apolipoprotein A-I (APOA1), 10 kDa heat shock protein (CH10, HSPE1), Annexin A2 (ANXA2), Myosin regulatory light polypeptide 9 (MYL9), Ras-related protein R-Ras (RRAS). E. Gene ontology (GO) analysis representing the protein class of proteins less abundant in TD colon. F. GO analysis of proteins more abundant in TD colon. G. Volcano plot visualizing proteins more abundant (in purple) and less abundant (in blue) in TD ovary. Proteins more abundant in TD ovary include Endoplasmic reticulum chaperone BiP (BiP), Plectin, Probable ATP-dependent RNA helicase (DDX17), collagens alpha-1(VI), alpha-2(VI), alpha-3(VI), alpha-1(XII), alpha-2(I), (CO6A1, CO6A2, CO6A3, COCA1, CO1A2), Tubulin beta-4B chain (TBB4B), Filamin-A (FLNA), Heat shock cognate 71 kDa protein (HSP7C), Immunoglobulin heavy constant gamma 1 (IGHG1), and Actin (ACTG). Proteins less abundant in TD ovary include RNA-binding protein FUS (FUS), Heterogeneous nuclear ribonucleoprotein K (HNRPK), 40S ribosomal protein S8 (RS8), Neuroblast differentiation-associated protein (AHNK), Y-box-binding protein 1 (YBOX1), RNA-binding motif protein (RBMX), Histone H2A type 1-J (H2A1J), Heterogeneous nuclear ribonucleoproteins C1/C2 (HNRPC). H. GO classes of proteins less abundant in TD ovary. I. Classes of proteins more abundant in TD ovary.

Figure 3

Post-MALDI IMS H&E stain of serial sections of nondenatured (A) compared to thermally denatured (B) human colon tissue. Ion images of peptides with m/z values 940.4621 (purple), 944.5420 (red), 976.4640 (yellow), and 1706.8124 (blue) in nondenatured (C) and TD (D) tissue. (E) Ion intensity plot of m/z 940.4621 shows increased detection in nondenatured tissue. (F) Ion intensity plot of m/z 944.5420 shows increased detection in TD tissue. (G) Ion intensity plot of m/z 976.4640 shows moderate increase in detection in TD tissue. (H) Ion intensity plot of m/z 1706.8124 showing dramatically improved detection in TD tissue.

Figure 4

Post-MALDI IMS H&E stain of serial sections of nondenatured (A) compared to thermally denatured (B) human ovary tissue. Ion images of peptides with m/z values 943.583 (blue), 957.590 (green), 980.450 (yellow), and 2790.337 (purple) in nondenatured (C) and TD (D) tissue. (E) Ion intensity plot of m/z 943.583 shows increased detection in nondenatured tissue. (F) Ion intensity plot of m/z 957.590 shows increased detection in TD tissue. (G) Ion intensity plot of m/z 980.450 shows increased detection TD tissue. (H) Ion intensity plot of m/z 2790.337 showing increased detection in TD tissue.

Figure 5

MALDI IMS ion images from nonthermally denatured (A,C,E) compared to thermally denatured (B,D,F) colon tissue and nonthermally denatured (G,I,K) compared to thermally denatured (H,J,L) ovary.