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Trapped ion mobility spectrometry (TIMS) using parallel accumulation serial fragmentation (PASEF ® ) is an advanced analytical technique that offers several advantages in mass spectrometry (MS)-based lipidomics. TIMS provides an additional dimension of separation to mass spectrometry and accurate collision cross-section (CCS) measurements for ions, aiding in the structural characterization of molecules. This is especially valuable in lipidomics for identifying and distinguishing isomeric or structurally similar compounds. On the other hand, PASEF technology allows for fast and efficient data acquisition by accumulating ions in parallel and then serially fragmenting them. This accelerates the analysis process and improves throughput, making it suitable for high-throughput applications. Moreover, the combination of TIMS and PASEF reduces co-elution and ion coalescence issues, leading to cleaner and more interpretable mass spectra. This results in higher data quality and more confident identifications. In this chapter, a data-dependent TIMS-PASEF ® workflow for lipidomics analysis is presented., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Molecular visualization of metabolites, lipids, and proteins by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is becoming an in-demand analytical approach to aid the histopathological analysis of breast cancer. Particularly, proteins seem to play a role in cancer progression, and specific proteins are currently used in the clinic for staging. Formalin-fixed paraffin-embedded (FFPE) tissues are ideal for correlating the molecular markers with clinical outcomes due to their long-term storage. So far, to obtain proteomic information by MSI from this kind of tissue, antigen retrieval and tryptic digestion steps are required. In this chapter, we present a protocol to spatially detect small proteins in tumor and necrotic regions of patient-derived breast cancer xenograft FFPE tissues without employing any on-tissue digestion. This protocol can be used for other kinds of FFPE tissue following specific optimization of the sample preparation phases., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry imaging (MSI) enables the spatial localization of proteins to be mapped directly on tissue sections, simultaneously detecting hundreds in a single analysis. However, the large data size, as well as the complexity of MALDI-MSI proteomics datasets, requires the appropriate tools and statistical approaches in order to reduce the complexity and mine the dataset in a successful manner. Here, a pipeline for the management of MALDI-MSI data is described, starting with preprocessing of the raw data, followed by statistical analysis using both supervised and unsupervised statistical approaches and, finally, annotation of those discriminatory protein signals highlighted by the data mining procedure.