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The application of innovative spatial proteomics techniques, such as those based upon matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technology, has the potential to impact research in the field of nephropathology. Notwithstanding, the possibility to apply this technology in more routine diagnostic contexts remains limited by the alternative fixatives employed by this ultraspecialized diagnostic field, where most nephropathology laboratories worldwide use bouin-fixed paraffin-embedded (BFPE) samples. Here, the feasibility of performing MALDI-MSI on BFPE renal tissue is explored, evaluating variability within the trypsin-digested proteome as a result of different preanalytical conditions and comparing them with the more standardized formalin-fixed paraffin-embedded (FFPE) counterparts. A large proportion of the features (270, 68.9%) was detected in both BFPE and FFPE renal samples, demonstrating only limited variability in signal intensity (10.22-10.06%). Samples processed with either fixative were able to discriminate the principal parenchyma regions along with diverse renal substructures, such as glomeruli, tubules, and vessels. This was observed when performing an additional "stress test", showing comparable results in both BFPE and FFPE samples when the distribution of several amyloid fingerprint proteins was mapped. These results suggest the utility of BFPE tissue specimens in MSI-based nephropathology research, further widening their application in this field.

Background: In the last decade the development of new PSMA-ligand based radiopharmaceuticals for the imaging and therapy of prostate cancer has been a highly active and important area of research. The most promising derivative in terms of interaction with the antigen and clinical properties has been found to be "PSMA-617", and its lutetium-177 radiolabelled version has recently been approved by EU and USA regulatory agencies for therapeutic purposes. For the above reasons, the development of new derivatives of PSMA-617 radiolabelled with fluorine-18 may still be of great interest. This paper proposes the comparison of two different PSMA-617 derivatives functionalized with NODA and RESCA chelators, respectively, radiolabelled via [18F]AlF2+ complexation. Results: The organic synthesis of two PSMA-617 derivatives and their radiolabelling via [18F]AlF2+ complexation resulted to proceed efficiently and successfully. Moreover, stability in solution and in plasma has been evaluated. The whole radiosynthesis procedure has been fully automated, and the final products have been obtained with radiochemical yield and purity potentially suitable for clinical studies. The biodistribution of the two derivatives was performed both in prostate cancer and glioma tumour models. Compared with the reference [18F]F-PSMA-1007 and [18F]F-PSMA-617-RESCA, [18F]F-PSMA-617-NODA derivative showed a higher uptake in both tumors, faster clearance in non-target organs, and lower uptake in salivary glands. Conclusion: PSMA-617 NODA and RESCA derivatives were radiolabelled successfully via [18F]AlF2+ chelation, the former being more stable in solution and human plasma. Moreover, preclinical biodistribution studies showed that [18F]F-PSMA-617-NODA might be of potential interest for clinical applications.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection leads to a wide range of clinical manifestations and determines the need for personalized and precision medicine. To better understand the biological determinants of this heterogeneity, we explored the plasma proteome of 43 COVID-19 patients with different outcomes by an untargeted liquid chromatography-mass spectrometry approach. The comparison between asymptomatic or pauci-symptomatic subjects (MILDs), and hospitalised patients in need of oxygen support therapy (SEVEREs) highlighted 29 proteins emerged as differentially expressed: 12 overexpressed in MILDs and 17 in SEVEREs. Moreover, a supervised analysis based on a decision-tree recognised three proteins (Fetuin-A, Ig lambda-2chain-C-region, Vitronectin) that are able to robustly discriminate between the two classes independently from the infection stage. In silico functional annotation of the 29 deregulated proteins pinpointed several functions possibly related to the severity; no pathway was associated exclusively to MILDs, while several only to SEVEREs, and some associated to both MILDs and SEVEREs; SARS-CoV-2 signalling pathway was significantly enriched by proteins up-expressed in SEVEREs (SAA1/2, CRP, HP, LRG1) and in MILDs (GSN, HRG). In conclusion, our analysis could provide key information for ‘proteomically’ defining possible upstream mechanisms and mediators triggering or limiting the domino effect of the immune-related response and characterizing severe exacerbations.

Ovarian cancer is a highly lethal gynecological malignancy. Drug resistance rapidly occurs, and different therapeutic approaches are needed. So far, no biomarkers have been discovered to predict early response to therapies in the case of multi-treated ovarian cancer patients. The aim of our investigation was to identify a protein panel and the molecular pathways involved in chemotherapy response through a combination of studying proteomics and network enrichment analysis by considering a subset of samples from a clinical setting. Differential mass spectrometry studies were performed on 14 serum samples from patients with heavily pretreated platinum-resistant ovarian cancer who received the FOLFOX-4 regimen as a salvage therapy. The serum was analyzed at baseline time (T0) before FOLFOX-4 treatment, and before the second cycle of treatment (T1), with the aim of understanding if it was possible, after a first treatment cycle, to detect significant proteome changes that could be associated with patients responses to therapy. A total of 291 shared expressed proteins was identified and 12 proteins were finally selected between patients who attained partial response or no-response to chemotherapy when both response to therapy and time dependence (T0, T1) were considered in the statistical analysis. The protein panel included APOL1, GSN, GFI1, LCATL, MNA, LYVE1, ROR1, SHBG, SOD3, TEC, VPS18, and ZNF573. Using a bioinformatics network enrichment approach and metanalysis study, relationships between serum and cellular proteins were identified. An analysis of protein networks was conducted and identified at least three biological processes with functional and therapeutic significance in ovarian cancer, including lipoproteins metabolic process, structural component modulation in relation to cellular apoptosis and autophagy, and cellular oxidative stress response. Five proteins were almost independent from the network (LYVE1, ROR1, TEC, GFI1, and ZNF573). All proteins were asso

Mass spectrometry imaging (MSI) is an emerging technology that is capable of mapping various biomolecules within their native spatial context, and performing spatial multiomics on formalin-fixed paraffin-embedded (FFPE) tissues may further increase the molecular characterization of pathological states. Here we present a novel workflow which enables the sequential MSI of lipids, N-glycans, and tryptic peptides on a single FFPE tissue section and highlight the enhanced molecular characterization that is offered by combining the multiple spatial omics data sets. In murine brain and clear cell renal cell carcinoma (ccRCC) tissue, the three molecular levels provided complementary information and characterized different histological regions. Moreover, when the spatial omics data was integrated, the different histopathological regions of the ccRCC tissue could be better discriminated with respect to the imaging data set of any single omics class. Taken together, these promising findings demonstrate the capability to more comprehensively map the molecular complexity within pathological tissue.

The podocyte injury, and consequent proteinuria, that characterize the pathology of idiopathic membranous nephropathy (IMN) is mediated by an autoimmune reaction against podocyte antigens. In particular, the activation of pathways leading to abundant renal deposits of complement is likely to involve the binding of mannose-binding lectin (MBL) to aberrant glycans on immunoglobulins. To obtain a landscape of circulatory IgG Fc glycosylation characterizing this disease, we conducted a systematic N-glycan profiling study of IgG1, 2, and 4 by mass spectrometry. The cohort included 57 IMN patients, a pathological control group with nephrotic syndrome (PN) (n = 20), and 88 healthy control subjects. The effect of sex and age was assessed in all groups and controlled by rigorous matching. Several IgG Fc glycan traits were found to be associated with IMN. Interestingly, among them, only IgG4-related results were specific for IMN and not for PN. Hypogalactosylation of IgG4, already shown for IMN, was observed to occur in the absence of core fucose, in line with a probable increase of pro-inflammatory IgG. In addition, elevated levels of fucosylated IgG4, along with low levels of hybrid-type glycans, were detected. Some of these IgG4 alterations are likely to be more pronounced in high PLA2R (phospholipase A2 receptor) patients. IgG Fc glycosylation patterns associated with IMN warrant further studies of their role in disease mechanisms and may eventually enrich the diagnostic spectrum regarding patient stratification.

The podocyte injury, and consequent proteinuria, that characterize the pathology of idiopathic membranous nephropathy (IMN) is mediated by an autoimmune reaction against podocyte antigens. In particular, the activation of pathways leading to abundant renal deposits of complement is likely to involve the binding of mannose-binding lectin (MBL) to aberrant glycans on immunoglobulins. To obtain a landscape of circulatory IgG Fc glycosylation characterizing this disease, we conducted a systematic N-glycan profiling study of IgG1, 2, and 4 by mass spectrometry. The cohort included 57 IMN patients, a pathological control group with nephrotic syndrome (PN) (n = 20), and 88 healthy control subjects. The effect of sex and age was assessed in all groups and controlled by rigorous matching. Several IgG Fc glycan traits were found to be associated with IMN. Interestingly, among them, only IgG4-related results were specific for IMN and not for PN. Hypogalactosylation of IgG4, already shown for IMN, was observed to occur in the absence of core fucose, in line with a probable increase of pro-inflammatory IgG. In addition, elevated levels of fucosylated IgG4, along with low levels of hybrid-type glycans, were detected. Some of these IgG4 alterations are likely to be more pronounced in high PLA2R (phospholipase A2 receptor) patients. IgG Fc glycosylation patterns associated with IMN warrant further studies of their role in disease mechanisms and may eventually enrich the diagnostic spectrum regarding patient stratification.

Since the start of the COVID-19 outbreak, more than four million people have died of this disease. Given its ability to provide a precise response, mass spectrometry-based proteomics could represent a useful tool to study this pathology. To this end, an untargeted nLC-ESI-MS/MS-based method to characterise SARS-CoV-2 proteins, including possible variants, and investigate human saliva and plasma proteome in a single analysis was developed for further application in patients. Four SARS-CoV-2 recombinant proteins, three (S1–S2–RBD) belonging to the spike glycoprotein (S) and one corresponding to the nucleoprotein (N), were prepared and analysed with nLC-UHRTOF by injecting decreasing amounts to establish the limit of detection (LOD) of the method. This was determined as 10 pg for all the components of the S protein and for N (71 amol and 213 amol, respectively). Various viral inactivation strategies plus deglycosylation and digestion approaches were then tested in saliva and plasma spiked with different quantities of SARS-CoV-2 recombinant proteins. The limit of characterisation (LOC) in saliva for the N and S proteins was observed at 100 pg (coverage of 20% and 3%, respectively); instead, in plasma, it was 33 pg for N and 330 pg for the S protein, with a coverage of 4% for both. About 300 and 800 human proteins were identified in plasma and saliva, respectively, including several key effectors and pathways that are known to be altered in COVID-19 patients. In conclusion, this approach allows SARS-CoV-2 proteins and the human proteome to be simultaneously explored, both for plasma and saliva, showing a high relevant potential for retrospective studies aimed at investigating possible virus variants and for patient stratification.