Sachdeva, Gunveen S.

ABSTRACT Imaging mass spectrometry has made significant advancements in recent years owing to its ability to study the spatial localization and abundance of proteins, peptides, lipids, glycans, metabolites, and drugs. In this study, we employed an imaging mass spectrometry‐based workflow called MALDI‐IHC, which uses antibodies conjugated with photocleavable mass‐tags, to investigate the detection of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) along with pan‐cytokeratin in formalin‐fixed paraffin‐embedded (FFPE) sections from cases with breast cancer. The expression of ER, PR, and HER2 was consistent with the clinical diagnosis based on conventional immunohistochemistry, lacking signal in triple‐negative cases and concordant signals in the cases that were classified as ER+/PR+/HER2+, ER+/PR+/HER2−, and ER−/PR−/HER2+ groups, respectively. In addition, the potential utility of a seconday antibody‐based MALDI‐IHC approach for HER2 expression was evaluated. In agreement with immunohistochemistry results, HER2 expression co‐localized within tumor regions in a HER2+ breast cancer case, while no expression was observed in a HER2− case. Collectively, these results highlight the potential of imaging mass spectrometry‐based MALDI‐IHC workflow for multiplexed detection of proteins in clinically relevant tissue sections.

Abstract Lewy pathology can form over decades in patients with Lewy body diseases, but the causal cellular mechanisms associated with this process remain unclear. This project aims to discover proteins that associate with monomeric and/or oligomeric alpha-synuclein during early stages of the aggregation process. To mimic the aggregation processes, cells expressing a synuclein-biotin ligase fusion protein were treated human recombinant pre-formed fibrils and subjected to BioSITe and mass spectrometry. Using a novel split biotin ligase fused to alpha-synuclein facilitated the identification of proteins specifically associated with multimeric alpha-synuclein. A total of 581 proteins were differentiated into potential interactors of monomeric versus multimeric alpha-synuclein in physiological versus aggregated conditions. The data reveal important phosphorylation mechanisms, connections to insulin processing, and a potential interaction with ALS-associated FUS. Interestingly, we identified that loss of specific interactions may contribute to pathology in patients with sporadic onset of Lewy body diseases. Future studies will validate both true interaction of highlighted proteins with alpha-synuclein, and the impact of such proteins on alpha-synuclein aggregation.

Key Points Increased protein expression of tissue inhibitor of metalloproteinase-3, glypican 6, synuclein γ , apolipoprotein A-4, and Ecto-5′-nucleotidase was observed in old adults compared with young adults. In old adults, overexpressed proteins were enriched in mitochondrial processes while under expressed proteins enriched in RNA metabolism. Validated differentially expressed proteins were not significantly correlated with kidney pathology after adjusting for age. Background Kidney aging is characterized by a loss of glomeruli, predominantly in the superficial cortex, with a resultant decline in GFR and an increased risk of various kidney-related diseases. The early molecular alterations in glomeruli associated with the aging process are not well studied. Methods We combined laser capture microdissection and mass spectrometry–based unbiased proteomic analysis of nonsclerosed, nonischemic glomeruli in the superficial cortex from young and old adults who underwent a radical nephrectomy for a tumor to understand the age-related molecular changes in glomeruli. Twenty-four young and 30 old adults were used for the discovery dataset, and the significant differentially expressed proteins were further validated using an independent set comprising six young and eight old adults. Results Kidneys from old adults had lower eGFR, less kidney parenchyma on computed tomography imaging, and more glomerulosclerosis and arteriosclerosis on histology. Quantitative proteomic analysis of nonsclerosed, nonischemic glomeruli identified increased expression of tissue inhibitor of metalloproteinase-3, glypican 6, synuclein γ , apolipoprotein A-4, and Ecto-5′-nucleotidase in old adults that were further validated in an independent set. Pathway analysis indicated that proteins with increased expression in old adults were enriched in mitochondrial translational processes, aerobic respiration, and TCA cycle, whereas proteins with decreased expression in old adults were enriched in mRNA splicing, mRNA processing, and nonsense mediated decay. Furthermore, validated differentially expressed proteins did not show any significant correlation with the kidney pathology independent of age group. Conclusions Overall, this study identified proteins that are specifically associated with the aging process in otherwise normal-appearing glomeruli.

BACKGROUND Diagnosis of PMM2-CDG, the most common congenital disorder of glycosylation (CDG), relies on measuring carbohydrate-deficient transferrin (CDT) and genetic testing. CDT tests have false negatives and may normalize with age. Site-specific changes in protein N-glycosylation have not been reported in sera in PMM2-CDG. METHODS Using multistep mass spectrometry–based N-glycoproteomics, we analyzed sera from 72 individuals to discover and validate glycopeptide alterations. We performed comprehensive tandem mass tag–based discovery experiments in well-characterized patients and controls. Next, we developed a method for rapid profiling of additional samples. Finally, targeted mass spectrometry was used for validation in an independent set of samples in a blinded fashion. RESULTS Of the 3,342 N-glycopeptides identified, patients exhibited decrease in complex-type N-glycans and increase in truncated, mannose-rich, and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man 5 GlcNAc 2 , which was not detected in controls, in 5 patients with normal CDT results, including 1 after liver transplant and 2 with a known genetic variant associated with mild disease, indicating greater sensitivity than CDT. It was detected by targeted analysis in 2 individuals with variants of uncertain significance in PMM2 . CONCLUSION Complement C4–derived Man 5 GlcNAc 2 glycopeptide could be a biomarker for accurate diagnosis and therapeutic monitoring of patients with PMM2-CDG and other CDGs. FUNDING U54NS115198 (Frontiers in Congenital Disorders of Glycosylation: NINDS; NCATS; Eunice Kennedy Shriver NICHD; Rare Disorders Consortium Disease Network); K08NS118119 (NINDS); Minnesota Partnership for Biotechnology and Medical Genomics; Rocket Fund; R01DK099551 (NIDDK); Mayo Clinic DERIVE Office; Mayo Clinic Center for Biomedical Discovery; IA/CRC/20/1/600002 (Center for Rare Disease Diagnosis, Research and Training; DBT/Wellcome Trust India Alliance)