In spatial’omics, highly confident molecular identifications are indispensable for the investigation of complex biology and for spatial biomarker discovery. However, current mass spectrometry imaging (MSI)-based spatial ‘omics must compromise between data acquisition speed and biochemical profiling depth. Here, we introduce fast, label-free quantum cascade laser mid-infrared imaging microscopy (QCL-MIR imaging) to guide MSI to high-interest tissue regions as small as kidney glomeruli, cultured multicellular spheroid cores or single motor neurons. Focusing on smaller tissue areas enables extensive spatial lipid identifications by on-tissue tandem-MS employing imaging parallel reaction monitoring-Parallel Accumulation-Serial Fragmentation (iprm-PASEF). QCL-MIR imaging-guided MSI allowed for unequivocal on-tissue elucidation of 157 sulfatides selectively accumulating in kidneys of arylsulfatase A-deficient mice used as ground truth concept and provided chemical rationales for improvements to ion mobility prediction algorithms. Using this workflow, we characterized sclerotic spinal cord lesions in mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, and identified upregulation of inflammation-related ceramide-1-phosphate and ceramide phosphatidylethanolamine as markers of white matter lipid remodeling. Taken together, widely applicable and fast QCL-MIR imaging-based guidance of MSI ensures that more time is available for exploration and validation of new biology by default on-tissue tandem-MS analysis.

Glycosphingolipids (GSLs) are important targets in immune, infectious, lysosomal storage diseases, cancer, and neurodegenerative diseases. Circulatory GSLs profiling in clinical samples is restricted by the lack of mid- and high-throughput analytical methods and deep coverage of long-chain sialylated glycosphingolipidome. We present a 4-dimensional (4D)-glycosphingolipidomics platform for routine glycosphingolipidome profiling encompassing: extraction and fractionation of sialylated GSLs with 3 to 15 monosaccharides, neutral GSLs and sulfatides; µL-flow reversed-phase LC-TIMS-PASEF MS analysis; semi-quantification strategy adapted for fractionated glycosphingolipidome, and referential CCS, RT, and m/z values for GSLs annotation. 4D-glycosphingolipidomics of human serum reveals a high structural heterogeneity, amounting to 376 GSLs: 159 GSLs of ganglio- and neolacto-series, 145 neutral GSLs and 72 sulfatides. Here we demonstrate the platform’s utility for clinical profiling of Parkinson’s disease (PD) sera. 41 neolacto- and ganglio-species discriminate PD patients from controls and 14 GSLs differentiate sex subgroups, laying the foundation for further functional GSL studies with PD.

In spatial ‘omics, highly confident molecular identifications are indispensable for the investigation of complex biology and for spatial biomarker discovery. However, current mass spectrometry (MS)-based spatial ‘omics must compromise between data acquisition speed and biochemical profiling depth, thus often leading to only “putative” molecular identifications. Here, we introduce fast quantum cascade laser mid-infrared imaging microscopy to guide MS imaging to confined tissue areas of high interest, e.g., multicellular spheroid cores or kidney glomeruli, for spatial lipidomics profiling at maximized analytical depth utilizing magnetic resonance-MS imaging at >10 ⁶ resolution or prm-PASEF-MS ² fragmentation imaging. Instigating selective sulfatide accumulation in arylsulfatase A-deficient mice as ground truth concept, we demonstrate that deep QCL-infrared-guided on-tissue spatial ‘omics unequivocally identifies 120 sulfatides. This approach enables identifications of odd-chain sulfatides and studies of structure-ion mobility-relationships that provide chemical rationales for improvements to current ion mobility prediction algorithms. Workflows and data processing tools are provided as community resources.

Background: Altered glycosphingolipidome in cancerous tissues and cells reflects the circulatory glycosphingolipid (GSL) profiles, which is advantageous for establishing cancer biomarkers and/or unravelling GSL-associated mechanisms of immunity in cancer. Methods: Here, we combined a microscale extraction of GSLs with multiple overlay TLC assays and IR-MALDI-o-TOF MS and implemented it for the first time in serum analysis of CD75s-, CD15s-, and iso-CD75s-containing sialylated GSLs of ganglio- and neolacto-series. Results: This sensitive antigen-specific targeted GSL workflow enabled the identification of 80 sialylated GSLs containing the specific antigens in human sera and was applied for the investigation of clinical serum samples from gastric/stomach cancer patients (n = 40), pancreatic cancer patients (n = 40), and a cancer-free control group (n = 20). The CD75s-, CD15s-, and iso-CD75s-containing GSL series encompassing complex monosialylated and fucosylated GSLs of neolacto-series, with up to pentadecasaccharide chains, were detected in both cancer types, while differential semi-quantitative analysis indicates a tumor type-specific associated GSL profile. Both cancer types share a drop in the complex fucosylated neolacto-gangliosides during tumor progression, implying a decreased synthesis of long-chain neolacto-series. Conclusions: This drop suggesting a role of these highly polar complex ganglioside species in evading humoral tumor immune response in the early tumor stages.