Protocol to analyze 1D and 2D mass spectrometry data from glioblastoma tissues for cancer diagnosis and immune cell identification.

In context of cancer diagnosis-based mass spectrometry (MS), the classification model created is crucial. Moreover, exploration of immune cell infiltration in tissues can offer insights within the tumor microenvironment. Here, we present a protocol to analyze 1D and 2D MS data from glioblastoma tissues for cancer diagnosis and immune cells identification. We describe steps for training the most optimal model and cross-validating it, for discovering robust biomarkers and obtaining their corresponding boxplots as well as creating an immunoscore based on MS-imaging data. For complete details on the use and execution of this protocol, please refer to Zirem et al.1.

Deciphering genetic and nongenetic factors underlying tumour dormancy: insights from multiomics analysis of two syngeneic MRD models of melanoma and leukemia.

BACKGROUND: Tumour dormancy, a resistance mechanism employed by cancer cells, is a significant challenge in cancer treatment, contributing to minimal residual disease (MRD) and potential relapse. Despite its clinical importance, the mechanisms underlying tumour dormancy and MRD remain unclear. In this study, we employed two syngeneic murine models of myeloid leukemia and melanoma to investigate the genetic, epigenetic, transcriptomic and protein signatures associated with tumour dormancy. We used a multiomics approach to elucidate the molecular mechanisms driving MRD and identify potential therapeutic targets. RESULTS: We conducted an in-depth omics analysis encompassing whole-exome sequencing (WES), copy number variation (CNV) analysis, chromatin immunoprecipitation followed by sequencing (ChIP-seq), transcriptome and proteome investigations. WES analysis revealed a modest overlap of gene mutations between melanoma and leukemia dormancy models, with a significant number of mutated genes found exclusively in dormant cells. These exclusive genetic signatures suggest selective pressure during MRD, potentially conferring resistance to the microenvironment or therapies. CNV, histone marks and transcriptomic gene expression signatures combined with Gene Ontology (GO) enrichment analysis highlighted the potential functional roles of the mutated genes, providing insights into the pathways associated with MRD. In addition, we compared "murine MRD genes" profiles to the corresponding human disease through public datasets and highlighted common features according to disease progression. Proteomic analysis combined with multi-omics genetic investigations, revealed a dysregulated proteins signature in dormant cells with minimal genetic mechanism involvement. Pathway enrichment analysis revealed the metabolic, differentiation and cytoskeletal remodeling processes involved in MRD. Finally, we identified 11 common proteins differentially expressed in dormant cells from both pathologies. CONCLUSIONS: Our study underscores the complexity of tumour dormancy, implicating both genetic and nongenetic factors. By comparing genomic, transcriptomic, proteomic, and epigenomic datasets, our study provides a comprehensive understanding of the molecular landscape of minimal residual disease. These results provide a robust foundation for forthcoming investigations and offer potential avenues for the advancement of targeted MRD therapies in leukemia and melanoma patients, emphasizing the importance of considering both genetic and nongenetic factors in treatment strategies.

In Vivo and Real-Time Metabolic Profiling of Plant-Microbe Interactions in Leaves, Stems, and Roots of Bacterially Inoculated Chardonnay Plantlets using SpiderMass.

There is growing interest in limiting the use of fungicides and implementing innovative, environmentally friendly strategies, such as the use of beneficial bacteria-triggered immunity, to protect grapevines from natural pathogens. Therefore, we need rapid and innovative ways to translate the knowledge of the molecular mechanisms underlying the activation of grapevine defenses against pathogens to induced resistance. Here, we have implemented an in vivo minimally invasive approach to study the interaction between plants and beneficial bacteria based on metabolic signatures. Paraburkholderia phytofirmans strain PsJN and PsJN-grapevine were used as bacterial and plant-bacterium interaction models, respectively. Using an innovative tool, SpiderMass, based on water-assisted laser desorption ionization with an IR microsampling probe, we simultaneously detect metabolic and lipidomic species. A metabolomic spectrum was thus generated, which was used to build a library and identify the most variable and discriminative peaks between the two conditions. We then showed that caftaric acid (m/z 311.04), caftaric acid dimer (m/z 623.09), derived caftaric acid (m/z 653.15), and quercetin-O-glucuronide tended to accumulate in grapevine leaves after root bacterization with PsJN. In addition, together with these phenolic messengers, we identified lipid biomarkers such as palmitic acid, linoleic acid, and α-linoleic acid as important messengers of enhanced defense mechanisms in Chardonnay plantlets. Taken together, SpiderMass is the next-generation methodology for studying plant-microorganism metabolic interactions with the prospect of in vivo real-time analysis in viticulture.

The impact and future of artificial intelligence in medical genetics and molecular medicine: an ongoing revolution.

Artificial intelligence (AI) platforms have emerged as pivotal tools in genetics and molecular medicine, as in many other fields. The growth in patient data, identification of new diseases and phenotypes, discovery of new intracellular pathways, availability of greater sets of omics data, and the need to continuously analyse them have led to the development of new AI platforms. AI continues to weave its way into the fabric of genetics with the potential to unlock new discoveries and enhance patient care. This technology is setting the stage for breakthroughs across various domains, including dysmorphology, rare hereditary diseases, cancers, clinical microbiomics, the investigation of zoonotic diseases, omics studies in all medical disciplines. AI's role in facilitating a deeper understanding of these areas heralds a new era of personalised medicine, where treatments and diagnoses are tailored to the individual's molecular features, offering a more precise approach to combating genetic or acquired disorders. The significance of these AI platforms is growing as they assist healthcare professionals in the diagnostic and treatment processes, marking a pivotal shift towards more informed, efficient, and effective medical practice. In this review, we will explore the range of AI tools available and show how they have become vital in various sectors of genomic research supporting clinical decisions.

Neuronal A2A receptor exacerbates synapse loss and memory deficits in APP/PS1 mice.

Early pathological upregulation of adenosine A2A receptors (A2ARs), one of the caffeine targets, by neurons is thought to be involved in the development of synaptic and memory deficits in Alzheimer's disease (AD) but mechanisms remain ill-defined. To tackle this question, we promoted a neuronal upregulation of A2AR in the hippocampus of APP/PS1 mice developing AD-like amyloidogenesis. Our findings revealed that the early upregulation of A2AR in the presence of an ongoing amyloid pathology exacerbates memory impairments of APP/PS1 mice. These behavioural changes were not linked to major change in the development of amyloid pathology but rather associated with increased phosphorylated tau at neuritic plaques. Moreover, proteomic and transcriptomic analyses coupled with quantitative immunofluorescence studies indicated that neuronal upregulation of the receptor promoted both neuronal and non-neuronal autonomous alterations, i.e. enhanced neuroinflammatory response but also loss of excitatory synapses and impaired neuronal mitochondrial function, presumably accounting for the detrimental effect on memory. Overall, our results provide compelling evidence that neuronal A2AR dysfunction, as seen in the brain of patients, contributes to amyloid-related pathogenesis and underscores the potential of A2AR as a relevant therapeutic target for mitigating cognitive impairments in this neurodegenerative disorder.

Tympanostomy Tubes Under Local Versus General Anesthesia for Children: A Prospective Long-Term Study.

OBJECTIVES: Tympanostomy tube insertion (TTI) under local anesthesia (LA) is gaining popularity but literature comparing long-term outcomes for children undergoing TTI under LA versus general anesthesia (GA) is limited. This study compares the long-term quality of life (QoL) between LA and GA in children undergoing TTI. Secondary objectives included long-term behavioral changes, parental satisfaction, tube durability, and postoperative complications. METHODS: We prospectively followed children aged under 6 who underwent TTI, under LA or GA, 2 years prior. We assessed QoL using validated scales (OM6, PedsQL), analyzed behavioral changes and parental satisfaction through qualitative scales, and retrieved data on tube durability and non-immediate complications. RESULTS: A total of 84 children (LA = 42; GA = 42) had complete data and a minimum of 1 year of follow-up. Demographic data were similar, except for younger patients in the LA group (1.4 vs. 1.9 years, p = 0.02). LA group exhibited increased fear of health care professionals following TTI (LA: Likert scale 2.1/5, GA: 1.5/5, p = 0.04). Tube retention rate was shorter in the LA group (at 15 months: GA:72%, LA:50%, p = 0.039). Two years post-TTI, there were no differences regarding QoL (OM-6 score; LA: 15.2/100, GA: 21.4/100, p = 0.18, and PedsQL score; LA: 84.3/100, GA: 83.8/100, p = 0.90), parental satisfaction with anesthesia (GA: 4.5/5, LA: 4.6/5, p = 0.56), and postoperative complications (GA: 3/42, LA: 7/42, p = 0.18). CONCLUSIONS: TTI under LA in children is associated with an increased fear of health care professionals and shorter functionality of tympanostomy tubes as compared to GA. No difference was observed in long-term QoL, parental satisfaction, and complications rate. LEVEL OF EVIDENCE: Level 3 Laryngoscope, 2024.

A co-culture system of macrophages with breast cancer tumoroids to study cell interactions and therapeutic responses.

3D tumoroids have revolutionized in vitro/ex vivo cancer biology by recapitulating the complex diversity of tumors. While tumoroids provide new insights into cancer development and treatment response, several limitations remain. As the tumor microenvironment, especially the immune system, strongly influences tumor development, the absence of immune cells in tumoroids may lead to inappropriate conclusions. Macrophages, key players in tumor progression, are particularly challenging to integrate into the tumoroids. In this study, we established three optimized and standardized methods for co-culturing human macrophages with breast cancer tumoroids: a semi-liquid model and two matrix-embedded models tailored for specific applications. We then tracked interactions and macrophage infiltration in these systems using flow cytometry and light sheet microscopy and showed that macrophages influenced not only tumoroid molecular profiles but also chemotherapy response. This underscores the importance of increasing the complexity of 3D models to more accurately reflect in vivo conditions.

Real-time glioblastoma tumor microenvironment assessment by SpiderMass for improved patient management.

Glioblastoma is a highly heterogeneous and infiltrative form of brain cancer associated with a poor outcome and limited therapeutic effectiveness. The extent of the surgery is related to survival. Reaching an accurate diagnosis and prognosis assessment by the time of the initial surgery is therefore paramount in the management of glioblastoma. To this end, we are studying the performance of SpiderMass, an ambient ionization mass spectrometry technology that can be used in vivo without invasiveness, coupled to our recently established artificial intelligence pipeline. We demonstrate that we can both stratify isocitrate dehydrogenase (IDH)-wild-type glioblastoma patients into molecular sub-groups and achieve an accurate diagnosis with over 90% accuracy after cross-validation. Interestingly, the developed method offers the same accuracy for prognosis. In addition, we are testing the potential of an immunoscoring strategy based on SpiderMass fingerprints, showing the association between prognosis and immune cell infiltration, to predict patient outcome.

Large Vestibular Aqueduct-Associated Symptoms: Endolymphatic Duct Blockage as a Surgical Treatment.

OBJECTIVE: This study aimed to evaluate the effectiveness of endolymphatic duct blockage (EDB) on dizziness control in patients with a large vestibular aqueduct (LVA) and to evaluate its effect on hearing. STUDY DESIGN: This is a prospective nonrandomized study. SETTING: Five adults and one child with dizziness and five children with progressive hearing loss were referred to our tertiary centers. METHODS: The dizziness handicap inventory (DHI) and DHI-PC (dizziness handicap inventory-patient caregiver) questionnaires were used before and after surgery. All patients underwent a preoperative temporal bone HRCT scan and pure tone audiometry one day before surgery, then four and twelve months after surgery and at the last follow-up. The mean follow-up time was 5.6 years. Student's t-test was used to compare DHI/-PC results. RESULTS: The DHI scores were 44, 24, 84, 59 and 56 before surgery, respectively, for Patients 1 to 5. The DHI scores at four months was significantly different, i.e., 4, 6, 0, 7 and 18 (p = 0.001). No differences were found between 4 and 12 months. Patient 6 (child) had Trisomy 21; their DHI-PC score dropped from 38 (preoperative score) to 8 (postoperative score), showing no activity limitations; clinical evaluation showed the complete resolution of symptoms. We found no significant differences between hearing loss before the surgery and at 1 and 12 months post operation for four adult patients. Our fifth adult patient's hearing changed from severe to profound SNHL. For 5 out of 6 pediatric patients, preoperative PTA and mean ABG were 63 dB and 20 dB, respectively; postoperatively, they improved to 42 dB and 16 dB, respectively. The hearing loss level for the sixth pediatric patient dropped from moderate (PTA = 42 dB) to severe (PTA = 85 dB) due to an opening of the endolymphatic sac and a sudden leak of the endolymph. CONCLUSIONS: EDB, using two titanium clips, seems to be helpful for controlling vestibular symptoms and for stabilizing hearing or even to improve hearing in 82% of cases. Nevertheless, there is a risk of hearing worsening.

The colibactin-producing Escherichia coli alters the tumor microenvironment to immunosuppressive lipid overload facilitating colorectal cancer progression and chemoresistance.

Intratumoral bacteria flexibly contribute to cellular and molecular tumor heterogeneity for supporting cancer recurrence through poorly understood mechanisms. Using spatial metabolomic profiling technologies and 16SrRNA sequencing, we herein report that right-sided colorectal tumors are predominantly populated with Colibactin-producing Escherichia coli (CoPEC) that are locally establishing a high-glycerophospholipid microenvironment with lowered immunogenicity. It coincided with a reduced infiltration of CD8+ T lymphocytes that produce the cytotoxic cytokines IFN-γ where invading bacteria have been geolocated. Mechanistically, the accumulation of lipid droplets in infected cancer cells relied on the production of colibactin as a measure to limit genotoxic stress to some extent. Such heightened phosphatidylcholine remodeling by the enzyme of the Land's cycle supplied CoPEC-infected cancer cells with sufficient energy for sustaining cell survival in response to chemotherapies. This accords with the lowered overall survival of colorectal patients at stage III-IV who were colonized by CoPEC when compared to patients at stage I-II. Accordingly, the sensitivity of CoPEC-infected cancer cells to chemotherapies was restored upon treatment with an acyl-CoA synthetase inhibitor. By contrast, such metabolic dysregulation leading to chemoresistance was not observed in human colon cancer cells that were infected with the mutant strain that did not produce colibactin (11G5∆ClbQ). This work revealed that CoPEC locally supports an energy trade-off lipid overload within tumors for lowering tumor immunogenicity. This may pave the way for improving chemoresistance and subsequently outcome of CRC patients who are colonized by CoPEC.

Comparison of Tympanostomy Tubes Under Local Anesthesia Versus General Anesthesia for Children.

OBJECTIVE: Tympanostomy tube insertion (TTI) is typically accomplished under general anesthesia (GA) in the operating room. We aimed to compare pain between GA and local anesthesia (LA) in surgically naïve children undergoing TTI. Secondary objectives examined patient's quality of life (QoL) and parent's satisfaction. STUDY DESIGN: Prospective single-center study. SETTING: Tertiary pediatric academic center. METHODS: Consecutive children who underwent TTI under GA were compared to patients under LA. Pain standardized observational pain scales (Face, Legs, Activity, Cry, Consolability Scale [FLACC], Children's hospital of Eastern Ontario Pain Scale [CHEOPS]) were completed pre-procedure, during the first tympanostomy and second tympanostomy, and post-procedure, as well as 1 week postoperatively. General health-related QoL (PedsQL) and QoL specific to otitis media (OM-6) were measured before insertion and 1 month postoperatively. Parental satisfaction was also evaluated using a qualitative scale. RESULTS: LA group had statistically significant higher pain levels at the beginning (7.3 vs. 0), during the first tympanostomy (7.8 vs. 0), during the second tympanostomy (7.7 vs. 0), and at end of the procedure (6.9 vs. 0) with the FLACC scale (all p < 0.01). Results were similar with the CHEOPS scale. No pain was noted 1 week after surgery in either group. Both groups had similar improvement in their QoL (p > 0.05). Minor complication occurred at a similar rate (p > 0.05). Parents were equally satisfied with their choice of anesthesia in both groups when initially questioned after the procedure (p > 0.05). CONCLUSIONS: Children experienced significantly less pain under GA than LA. If LA is to be used, pain and distress-reducing strategies are critical. Shared decision-making with families is essential. LEVEL OF EVIDENCE: 3 Laryngoscope, 134:2422-2429, 2024.

OpenProt 2.0 builds a path to the functional characterization of alternative proteins.

The OpenProt proteogenomic resource (https://www.openprot.org/) provides users with a complete and freely accessible set of non-canonical or alternative open reading frames (AltORFs) within the transcriptome of various species, as well as functional annotations of the corresponding protein sequences not found in standard databases. Enhancements in this update are largely the result of user feedback and include the prediction of structure, subcellular localization, and intrinsic disorder, using cutting-edge algorithms based on machine learning techniques. The mass spectrometry pipeline now integrates a machine learning-based peptide rescoring method to improve peptide identification. We continue to help users explore this cryptic proteome by providing OpenCustomDB, a tool that enables users to build their own customized protein databases, and OpenVar, a genomic annotator including genetic variants within AltORFs and protein sequences. A new interface improves the visualization of all functional annotations, including a spectral viewer and the prediction of multicoding genes. All data on OpenProt are freely available and downloadable. Overall, OpenProt continues to establish itself as an important resource for the exploration and study of new proteins.

Comparing MS imaging of lipids by WALDI and MALDI: two technologies for evaluating a common ground truth in MS imaging.

In this study, we conducted a direct comparison of water-assisted laser desorption ionization (WALDI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging, with MALDI serving as the benchmark for label-free molecular tissue analysis in biomedical research. Specifically, we investigated the lipidomic profiles of several biological samples and calculated the similarity of detected peaks and Pearson's correlation of spectral profile intensities between the two techniques. We show that, overall, MALDI MS and WALDI MS present very close lipidomic analyses and that the highest similarity is obtained for the norharmane MALDI matrix. Indeed, for norharmane in negative ion mode, the lipidomic spectra revealed 100% similarity of detected peaks and over 0.90 intensity correlation between both technologies for five samples. The MALDI-MSI positive ion lipid spectra displayed more than 83% similarity of detected peaks compared to those of WALDI-MSI. However, we observed a lower percentage (77%) of detected peaks when comparing WALDI-MSI with MALDI-MSI due to the rich WALDI-MSI lipid spectra. Despite this difference, the global lipidomic spectra showed high consistency between the two technologies, indicating that they are governed by similar processes. Thanks to this similarity, we can increase datasets by including data from both modalities to either co-train classification models or obtain cross-interrogation.

Fallopian tube lesions as potential precursors of early ovarian cancer: a comprehensive proteomic analysis.

Ovarian cancer is the leading cause of death from gynecologic cancer worldwide. High-grade serous carcinoma (HGSC) is the most common and deadliest subtype of ovarian cancer. While the origin of ovarian tumors is still debated, it has been suggested that HGSC originates from cells in the fallopian tube epithelium (FTE), specifically the epithelial cells in the region of the tubal-peritoneal junction. Three main lesions, p53 signatures, STILs, and STICs, have been defined based on the immunohistochemistry (IHC) pattern of p53 and Ki67 markers and the architectural alterations of the cells, using the Sectioning and Extensively Examining the Fimbriated End Protocol. In this study, we performed an in-depth proteomic analysis of these pre-neoplastic epithelial lesions guided by mass spectrometry imaging and IHC. We evaluated specific markers related to each preneoplastic lesion. The study identified specific lesion markers, such as CAVIN1, Emilin2, and FBLN5. We also used SpiderMass technology to perform a lipidomic analysis and identified the specific presence of specific lipids signature including dietary Fatty acids precursors in lesions. Our study provides new insights into the molecular mechanisms underlying the progression of ovarian cancer and confirms the fimbria origin of HGSC.

Heimdall, an alternative protein issued from a ncRNA related to kappa light chain variable region of immunoglobulins from astrocytes: a new player in neural proteome.

The dogma "One gene, one protein" is clearly obsolete since cells use alternative splicing and generate multiple transcripts which are translated into protein isoforms, but also use alternative translation initiation sites (TISs) and termination sites on a given transcript. Alternative open reading frames for individual transcripts give proteins originate from the 5'- and 3'-UTR mRNA regions, frameshifts of mRNA ORFs or from non-coding RNAs. Longtime considered as non-coding, recent in-silico translation prediction methods enriched the protein databases allowing the identification of new target structures that have not been identified previously. To gain insight into the role of these newly identified alternative proteins in the regulation of cellular functions, it is crucial to assess their dynamic modulation within a framework of altered physiological modifications such as experimental spinal cord injury (SCI). Here, we carried out a longitudinal proteomic study on rat SCI from 12 h to 10 days. Based on the alternative protein predictions, it was possible to identify a plethora of newly predicted protein hits. Among these proteins, some presented a special interest due to high homology with variable chain regions of immunoglobulins. We focus our interest on the one related to Kappa variable light chains which is similarly highly produced by B cells in the Bence jones disease, but here expressed in astrocytes. This protein, name Heimdall is an Intrinsically disordered protein which is secreted under inflammatory conditions. Immunoprecipitation experiments showed that the Heimdall interactome contained proteins related to astrocyte fate keepers such as "NOTCH1, EPHA3, IPO13" as well as membrane receptor protein including "CHRNA9; TGFBR, EPHB6, and TRAM". However, when Heimdall protein was neutralized utilizing a specific antibody or its gene knocked out by CRISPR-Cas9, sprouting elongations were observed in the corresponding astrocytes. Interestingly, depolarization assays and intracellular calcium measurements in Heimdall KO, established a depolarization effect on astrocyte membranes KO cells were more likely that the one found in neuroprogenitors. Proteomic analyses performed under injury conditions or under lipopolysaccharides (LPS) stimulation, revealed the expression of neuronal factors, stem cell proteins, proliferation, and neurogenesis of astrocyte convertor factors such as EPHA4, NOTCH2, SLIT3, SEMA3F, suggesting a role of Heimdall could regulate astrocytic fate. Taken together, Heimdall could be a novel member of the gatekeeping astrocyte-to-neuroprogenitor conversion factors.

Development of a novel apigenin prodrug programmed for alkaline-phosphatase instructed self-inhibition to combat cancer.

Elevated levels of alkaline phosphatase (ALP) in the tumor microenvironment (TME) are a hallmark of cancer progression and thus inhibition of ALP could serve as an effective approach against cancer. Herein, we developed a novel prodrug approach to tackle cancer that bears self-inhibiting alkaline phosphatase-responsiveness properties that can enhance at the same time the solubility of the parent compound. To probe this novel concept, we selected apigenin as the cytotoxic agent since we first unveiled, that it directly interacts and inhibits ALP activity. Consequently, we rationally designed and synthesized, using a self-immolative linker, an ALP responsive apigenin-based phosphate prodrug, phospho-apigenin. Phospho-apigenin markedly increased the stability of the parent compound apigenin. Furthermore, the prodrug exhibited enhanced antiproliferative effect in malignant cells with elevated ALP levels, compared to apigenin. This recorded potency of the developed prodrug was further confirmed in vivo where phospho-apigenin significantly suppressed by 52.8% the growth of PC-3 xenograft tumors.Communicated by Ramaswamy H. Sarma.

Early Diagnosis: End-to-End CNN-LSTM Models for Mass Spectrometry Data Classification.

Liquid chromatography-mass spectrometry (LC-MS) is a powerful method for cell profiling. The use of LC-MS technology is a tool of choice for cancer research since it provides molecular fingerprints of analyzed tissues. However, the ubiquitous presence of noise, the peaks shift between acquisitions, and the huge amount of information owing to the high dimensionality of the data make rapid and accurate cancer diagnosis a challenging task. Deep learning (DL) models are not only effective classifiers but are also well suited to jointly learn feature representation and classification tasks. This is particularly relevant when applied to raw LC-MS data and hence avoid the need for costly preprocessing and complicated feature selection. In this study, we propose a new end-to-end DL methodology that addresses all of the above challenges at once, while preserving the high potential of LC-MS data. Our DL model is designed to early discriminate between tumoral and normal tissues. It is a combination of a convolutional neural network (CNN) and a long short-term memory (LSTM) Network. The CNN network allows for significantly reducing the high dimensionality of the data while learning spatially relevant features. The LSTM network enables our model to capture temporal patterns. We show that our model outperforms not only benchmark models but also state-of-the-art models developed on the same data. Our framework is a promising strategy for improving early cancer detection during a diagnostic process.

Ambient Mass Spectrometry Imaging by Water-Assisted Laser Desorption/Ionization for Ex Vivo and in Vivo Applications.

Water-assisted laser desorption/ionization mass spectrometry (WALDI-MS), also known as SpiderMass, is an emerging ambient ionization technique for in vivo and real-time analysis. It employs a remote infrared (IR) laser tuned to excite the most intense vibrational band (O-H) of water. The water molecules act as an endogenous matrix leading to the desorption/ionization of a variety of biomolecules from tissues, particularly metabolites and lipids. WALDI-MS was recently advanced into an imaging modality for ex vivo 2D sections and 3D in vivo real-time imaging. Here, we describe the methodological aspects for performing 2D and 3D imaging experiments with WALDI-MSI and the parameters for optimizing the image acquisition.

Protocol to identify human subcellular alternative protein interactions using cross-linking mass spectrometry.

Since the start of mass-spectrometry-based proteomics, proteins from non-referenced open reading frames or alternative proteins (AltProts) have been overlooked. Here, we present a protocol to identify human subcellular AltProt and decipher some interactions using cross-linking mass spectrometry. We describe steps for cell culture, in cellulo cross-link, subcellular extraction, and sequential digestion. We then detail both liquid chromatography-tandem mass spectrometry and cross-link data analyses. The implementation of a single workflow allows the non-targeted identification of signaling pathways involving AltProts. For complete details on the use and execution of this protocol, please refer to Garcia-del Rio et al.1.