Cintron-Diaz Yarixa L, Gomez-Hernandez Mario E, Verhaert Marthe M H A, Verhaert Peter D E M, Fernandez-Lima Francisco
Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, AHC4-233, Miami, Florida 33199, United States.
ProteoFormiX, JLABS@BE, Janssen Pharmaceutica Campus, Turnhoutseweg 30, B-2340 Beerse, Belgium.
J Am Soc Mass Spectrom. 2022 Apr 6;33(4):681-687. doi: 10.1021/jasms.1c00376. Epub 2022 Mar 8.
To make the vast collections of well-documented human clinical samples archived in biobanks accessible for mass spectrometry imaging (MSI), recent developments have focused on the label-free top-down MS analysis of neuropeptides in sections of formalin-fixed, paraffin-embedded (FFPE) tissues. In analogy to immunohistochemistry (IHC), this variant of MSI has been designated MSHC (mass spectrometry histochemistry). Besides the detection and localization of neuropeptide and other biomolecular MS signals in these FFPE samples, there is great interest in their molecular identification and full characterization. We here used matrix assisted laser desorption ionization (MALDI) MSI employing ultrahigh-resolution FT-ICR MS on 2,5-dihydroxybenzoic acid (DHB) coated five-micron sections of human FFPE pituitary to demonstrate clear isotope patterns and elemental composition assignment of neuropeptides (with ∼1 ppm mass accuracy). Besides tandem MS fragmentation pattern analysis to deduce or confirm amino acid sequence information (Arg-vasopressin for the case presented here), there is a need for orthogonal primary structure characterization of the peptide-like MS signals of biomolecules desorbed directly off FFPE tissue sections. In the present work, we performed liquid extraction surface analysis (LESA) extractions on consecutive (uncoated) tissue slices. This enables the successful characterization by ion mobility MS of vasopressin present in FFPE material. Differences in sequence coverage are discussed on the basis of the mobility selected collision induced dissociation (CID), electron capture dissociation (ECD), and UV photodissociation (UVPD) MS/MS. Using Arg-vasopressin as model case (a peptide with a disulfide bridged ring structure), we illustrate the use of LESA in combination with a reduction agent for effective sequencing using mobility selected CID, ECD, and UVPD MS/MS.
为了使生物样本库中存档的大量记录完备的人类临床样本能够用于质谱成像（MSI），近期的研究进展主要集中在对福尔马林固定、石蜡包埋（FFPE）组织切片中的神经肽进行无标记的自上而下的质谱分析。类似于免疫组织化学（IHC），这种MSI变体被称为MSHC（质谱组织化学）。除了在这些FFPE样本中检测和定位神经肽及其他生物分子的质谱信号外，人们对其分子鉴定和全面表征也有着浓厚的兴趣。我们在此使用基质辅助激光解吸电离（MALDI）MSI，在涂有2,5 - 二羟基苯甲酸（DHB）的人类FFPE垂体五微米切片上采用超高分辨率傅里叶变换离子回旋共振质谱（FT-ICR MS），以展示神经肽清晰的同位素模式和元素组成归属（质量精度约为1 ppm）。除了进行串联质谱碎片模式分析以推导或确认氨基酸序列信息（此处以精氨酸加压素为例）外，还需要对直接从FFPE组织切片解吸的生物分子的肽样质谱信号进行正交一级结构表征。在本工作中，我们对连续的（未涂层的）组织切片进行了液相萃取表面分析（LESA）萃取。这使得能够通过离子淌度质谱成功表征FFPE材料中存在的加压素。基于淌度选择的碰撞诱导解离（CID）、电子捕获解离（ECD）和紫外光解离（UVPD）串联质谱，讨论了序列覆盖的差异。以精氨酸加压素作为模型案例（一种具有二硫键桥接环结构的肽），我们说明了LESA与还原剂结合使用，通过淌度选择的CID、ECD和UVPD串联质谱进行有效测序的应用。
