Sublimation of new matrix candidates for high spatial resolution imaging mass spectrometry of lipids: enhanced information in both positive and negative polarities after 1,5-diaminonapthalene deposition.

Matrix sublimation has demonstrated to be a powerful approach for high-resolution matrix-assisted laser desorption ionization (MALDI) imaging of lipids, providing very homogeneous solvent-free deposition. This work presents a comprehensive study aiming to evaluate current and novel matrix candidates for high spatial resolution MALDI imaging mass spectrometry of lipids from tissue section after deposition by sublimation. For this purpose, 12 matrices including 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,6-dihydroxyacetphenone (DHA), 2',4',6'-trihydroxyacetophenone (THAP), 3-hydroxypicolinic acid (3-HPA), 1,8-bis(dimethylamino)naphthalene (DMAN), 1,8,9-anthracentriol (DIT), 1,5-diaminonapthalene (DAN), p-nitroaniline (NIT), 9-aminoacridine (9-AA), and 2-mercaptobenzothiazole (MBT) were investigated for lipid detection efficiency in both positive and negative ionization modes, matrix interferences, and stability under vacuum. For the most relevant matrices, ion maps of the different lipid species were obtained from tissue sections at high spatial resolution and the detected peaks were characterized by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. First proposed for imaging mass spectrometry (IMS) after sublimation, DAN has demonstrated to be of high efficiency providing rich lipid signatures in both positive and negative polarities with high vacuum stability and sub-20 µm resolution capacity. Ion images from adult mouse brain were generated with a 10 µm scanning resolution. Furthermore, ion images from adult mouse brain and whole-body fish tissue sections were also acquired in both polarity modes from the same tissue section at 100 µm spatial resolution. Sublimation of DAN represents an interesting approach to improve information with respect to currently employed matrices providing a deeper analysis of the lipidome by IMS.

Novel homozygous nonsense mutation of MLIP and compensatory alternative splicing.

Despite the growing accessibility of clinical sequencing, functional interpretation of variants remains a major hurdle to molecular diagnostics of Mendelian diseases. We aimed to describe a new adult-onset myopathy with muscle weakness and hyperCKemia caused by a nonsense variant in muscular LMNA-interacting protein (MLIP). Following RNA-sequencing, differential expression analysis uncovered a significant downregulation of this gene, which had a surprisingly mild effect on MLIP protein expression. RT-PCR and long-read sequencing (LRS) both support an important transcriptome shift in the patient, where decreased MLIP levels are seemingly due to nonsense-mediated decay of transcripts containing the exon 5 mutation. Moreover, a compensatory mechanism upregulates the functionally lacking isoforms and generates novel transcripts. These results support the recently discovered clinical implications of MLIP variants in myopathies, highlighting for the first time its relevance in adult-onset cases. These results also underline the power of LRS as a tool for the functional assessment of variants of unknown significance (VUS), as well as the definition of accurate isoform profile annotations in a tissue-specific manner.

Orthogonal organic and aqueous-based washes of tissue sections to enhance protein sensitivity by MALDI imaging mass spectrometry.

Imaging mass spectrometry (IMS) is an emergent and innovative approach for measuring the composition, abundance and regioselectivity of molecules within an investigated area of fixed dimension. Although providing unprecedented molecular information compared with conventional MS techniques, enhancement of protein signature by IMS is still necessary and challenging. This paper demonstrates the combination of conventional organic washes with an optimized aqueous-based buffer for tissue section preparation before matrix-assisted laser desorption/ionization (MALDI) IMS of proteins. Based on a 500 mM ammonium formate in water-acetonitrile (9:1; v/v, 0.1% trifluororacetic acid, 0.1% Triton) solution, this buffer wash has shown to significantly enhance protein signature by profiling and IMS (~fourfold) when used after organic washes (70% EtOH followed by 90% EtOH), improving the quality and number of ion images obtained from mouse kidney and a 14-day mouse fetus whole-body tissue sections, while maintaining a similar reproducibility with conventional tissue rinsing. Even if some protein losses were observed, the data mining has demonstrated that it was primarily low abundant signals and that the number of new peaks found is greater with the described procedure. The proposed buffer has thus demonstrated to be of high efficiency for tissue section preparation providing novel and complementary information for direct on-tissue MALDI analysis compared with solely conventional organic rinsing.