Mass spectrometry imaging protocol for spatial mapping of lipids, N-glycans and peptides in murine lung tissue.

RATIONALE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Performance comparison of homologous and heterologous Newcastle disease virus in vaccines and antibody tests.

Antigenic differences between commercial Newcastle Disease Virus (NDV) vaccine and circulating field virus reduce vaccine efficacy. Fifty-layer chickens were divided into five groups: three vaccinated chicken groups using killed LaSota (Genotype II/GII), Mega, or VD (Genotype VII/GVII) viral strains, negative, and positive control groups. On day 28, Hemagglutination Inhibition (HI) serology of vaccinated chickens was performed using whole virus antigens of RIVS, LaSota, Mega, and VD strains. Sera were also tested with an alternative antigen, using an ELISA to detect antibody for the cleavage site F protein peptide from GII and GVII NDV strains. Vaccinated and unvaccinated positive control birds underwent infectious challenges using VD and Mega strains. HI testing showed that antibody titers were higher when tested using homologous antigens than heterologous antigens. ELISA performed with alternative antigens did not perform as well as the established HI test using homologous strains. Viral shedding was reduced by vaccination that was homologous to the infectious challenge in comparison with vaccination using the LaSota strain virus. We conclude that superior results are obtained when serological testing, vaccinations, and vaccine challenge experiments all use circulating strains of ND virus. Implementation of this recommendation would likely reduce viral shedding by vaccinated chickens and be more effective in preventing outbreaks of virulent NDV.