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

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.

热变性（Thermal denaturation，TD）又称抗原修复（antigen retrieval），指将组织样本置于缓冲溶液中加热以暴露蛋白质表位。福尔马林固定石蜡包埋样本的热变性可增强组织原位胰蛋白酶消化，提升基质辅助激光解吸电离成像质谱（matrix-assisted laser desorption ionization imaging mass spectrometry，MALDI IMS）对肽段的检测效能。本研究针对未固定的冷冻人结肠、卵巢及胰腺组织，探究了TD对肽段MALDI IMS及液相色谱-串联质谱信号的组织依赖性影响。本研究采用飞行时间（time-of-flight，TOF）、轨道阱（orbitrap）及傅里叶变换离子回旋共振质谱（Fourier-transform ion cyclotron resonance mass spectrometry）平台开展三次重复实验，结果显示TD对肽段信号的影响具有组织特异性：结肠组织的肽段检测率提升22.5%，卵巢组织提升73.3%，胰腺组织则提升96.6%。对已鉴定肽段的生化分析表明，TD可促进疏水肽段的鉴定。