Clinical & Health Sciences, University of South Australia, Adelaide, SA, Australia.
Rapid Commun Mass Spectrom. 2024 May 15;38(9):e9721. doi: 10.1002/rcm.9721.
The application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to murine lungs is challenging due to the spongy nature of the tissue. Lungs consist of interconnected air sacs (alveoli) lined by a single layer of flattened epithelial cells, which requires inflation to maintain its natural structure. Therefore, a protocol that is compatible with both lung instillation and high spatial resolution is essential to enable multi-omic studies on murine lung disease models using MALDI-MSI.
To maintain the structural integrity of the tissue, murine lungs were inflated with 8% (w/v) gelatin for lipid MSI of fresh frozen tissues or 4% (v/v) paraformaldehyde neutral buffer for N-glycan and peptide MSI of FFPE tissues. Tissues were sectioned and prepared for enzymatic digestion and/or matrix deposition. Glycerol-free PNGase F was applied for N-glycan MSI, while Trypsin Gold was applied for peptide MSI using the iMatrixSpray and ImagePrep Station, respectively. For lipid, N-glycan and peptide MSI, α-cyano-4-hydroxycinnamic acid matrix was deposited using the iMatrixSpray. MS data were acquired with 20 μm spatial resolution using a timsTOF fleX MS instrument followed by MS fragmentation of lipids, N-glycans and peptides. For lipid MSI, trapped ion mobility spectrometry was used to separate isomeric/isobaric lipid species. SCiLS™ Lab was used to visualize all MSI data. For analyte identification, MetaboScape®, GlycoMod and Mascot were used to annotate MS fragmentation spectra of lipids, N-glycans and tryptic peptides, respectively.
Our protocol provides instructions on sample preparation for high spatial resolution MALDI-MSI, MS/MS data acquisition and lipid, N-glycan and peptide annotation and identification from murine lungs. This protocol will allow non-biased analyses of diseased lungs from preclinical murine models and provide further insight into disease models.
由于组织的海绵状性质，将基质辅助激光解吸/电离质谱成像（MALDI-MSI）应用于鼠肺具有挑战性。肺由相互连接的气囊（肺泡）组成，由单层扁平上皮细胞排列，需要充气来维持其天然结构。因此，需要一种既兼容肺灌注又兼容高空间分辨率的方案，以便使用 MALDI-MSI 对鼠肺疾病模型进行多组学研究。
为了保持组织的结构完整性，用 8%（w/v）明胶对鼠肺充气，用于新鲜冷冻组织的脂质 MALDI-MSI，或用 4%（v/v）多聚甲醛中性缓冲液对 FFPE 组织的 N-糖链和肽 MALDI-MSI。对组织进行切片并准备进行酶消化和/或基质沉积。应用无甘油 PNGase F 进行 N-糖链 MALDI-MSI，而应用胰蛋白酶 Gold 分别通过 iMatrixSpray 和 ImagePrep Station 进行肽 MALDI-MSI。对于脂质、N-糖链和肽 MALDI-MSI，用 iMatrixSpray 沉积α-氰基-4-羟基肉桂酸基质。使用 timsTOF fleX MS 仪器以 20μm 空间分辨率采集 MS 数据，然后对脂质、N-糖链和肽进行 MS 碎裂。对于脂质 MALDI-MSI，使用俘获离子淌度质谱法分离异构/同重质脂质。使用 SCiLS™ Lab 可视化所有 MALDI-MSI 数据。对于分析物鉴定，分别使用 MetaboScape®、GlycoMod 和 Mascot 注释脂质、N-糖链和胰蛋白酶肽的 MS 碎裂谱。
本方案提供了高空间分辨率 MALDI-MSI 样品制备、MS/MS 数据采集以及从鼠肺中脂质、N-糖链和肽注释和鉴定的说明。该方案将允许对临床前鼠模型的患病肺进行无偏分析，并为疾病模型提供更深入的了解。
