Maastricht MultiModal Molecular Imaging (M4I) Institute, Division of Imaging Mass Spectrometry , Maastricht University , Universiteitssingel 50 , 6229ER Maastricht , The Netherlands.
Bioinformatics Solutions Inc. , 470 Weber Street North , Waterloo , Ontario N2L 6J2 , Canada.
Anal Chem. 2018 Aug 7;90(15):9272-9280. doi: 10.1021/acs.analchem.8b01838. Epub 2018 Jul 18.
Formalin-fixed neuroendocrine tissues from American cockroaches ( Periplaneta americana) embedded in paraffin more than 30 years ago were recently analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), to reveal the histological localization of more than 20 peptide ions. These represented protonated, and other cationic species of, at least, 14 known neuropeptides. The characterization of peptides in such historical samples was made possible by a novel sample preparation protocol rendering the endogenous peptides readily amenable to MSI analysis. The protocol comprises brief deparaffinization steps involving xylene and ethanol, and is further devoid of conventional aqueous washing, buffer incubations, or antigen retrieval steps. Endogenous secretory peptides that are typically highly soluble are therefore retained in-tissue with this protocol. The method is fully "top-down", that is, without laborious in situ enzymatic digestion that typically disturbs the detection of low-abundance endogenous peptides by MSI. Peptide identifications were supported by accurate mass, on-tissue tandem MS analyses, and by earlier MALDI-MSI results reported for freshly prepared P. americana samples. In contrast to earlier literature accounts stating that MALDI-MSI detection of endogenous peptides is possible only in fresh or freshly frozen tissues, or exceptionally, in formalin-fixed, paraffin-embedded (FFPE) material of less than 1 year old, we demonstrate that MALDI-MSI works for endogenous peptides in FFPE tissue of up to 30 years old. Our findings put forward a useful method for digestion-free, high-throughput analysis of endogenous peptides from FFPE samples and offer the potential for reinvestigating archived and historically interesting FFPE material, such as those stored in hospital biobanks.
三十多年前嵌入石蜡的美洲大蠊（Periplaneta americana）固定组织，最近通过基质辅助激光解吸/电离质谱成像（MALDI-MSI）进行了分析，以揭示 20 多种肽离子的组织学定位。这些肽代表至少 14 种已知神经肽的质子化和其他阳离子物种。这种历史样本中的肽的特征是通过一种新的样本制备方案实现的，该方案使内源性肽易于进行 MSI 分析。该方案包括涉及二甲苯和乙醇的简短脱蜡步骤，并且进一步没有传统的水性洗涤、缓冲液孵育或抗原回收步骤。因此，与传统方法相比，通常高度溶解的内源性分泌肽可以保留在组织中。该方法完全是“自上而下”的，也就是说，无需费力的原位酶消化，因为这通常会干扰 MSI 对低丰度内源性肽的检测。肽鉴定得到精确质量、组织上串联 MS 分析以及对新鲜制备的美洲大蠊样本的早期 MALDI-MSI 结果的支持。与早期文献报道的 MALDI-MSI 仅能检测新鲜或新鲜冷冻组织中内源性肽，或者特别地，在不到 1 年的福尔马林固定、石蜡包埋（FFPE）材料中检测内源性肽的说法相反，我们证明 MALDI-MSI 可用于 30 年内的 FFPE 组织中内源性肽的检测。我们的发现提出了一种从 FFPE 样本中进行无消化、高通量分析内源性肽的有用方法，并为重新研究存档的和历史上有趣的 FFPE 材料（如存储在医院生物库中的材料）提供了潜力。
