Quantitative Chemoproteomic Analysis of Protein O-GlcNAcylation
Abstract
O-GlcNAc (O-linked N-acetylglucosamine) modification is a non-canonical form of protein glycosylation, which occurs intracellular and is dynamically regulated. In mammalian cells, more than a thousand cytoplasmic, nuclear, and mitochontrial proteins are post-translationally modified with O-GlcNAc, which regulates many important biological processes. OGT and OGA are two enzymes responsible for dynamically cycling O-GlcNAc on and off the modified proteins. How O-GlcNAc affects protein stability remains to be investigated at the proteome level. In this talk, I will present the development of a time-resolved quantitative proteomic strategy to analyze the turnover dynamics of O-GlcNAcylated proteins. We found that not all protein O-GlcNAcylation events were reversible. Interestingly, a subset of O-GlcNAcylated proteins are hyper-stable, exhibiting minimal removal of O-GlcNAc or degradation of protein backbones. The hyper-stable population included three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs), fibrillarin (FBL), NOP56, and NOP58. Our studies showed that O-GlcNAcylation stabilized these proteins and regulated snoRNP assembly. Blocking O-GlcNAcylation on FBL altered the 2’-O-methylation of ribosomal RNAs, and impaired cancer cell proliferation and tumor formation in vivo. Our work reveals stable O-GlcNAc as an important regulatory mechanism for stabilizing proteins.
Supramolecular polymorphism in Pathogenic Amyloid Proteins by Cu(II) Coordination
Abstract
Current understandings in synucleinopathies propose that α-synuclein (αSyn) aggregates with different fibrillar structures appear to have distinct pathogenic characteristics in cell-transmissibility and neurotoxicity. However, the origination of structural polymorphism in αSyn fibrils remains largely unknown. In this presentation, I will discuss divalent metal interactions of Syn replated to its polymorphisms and cell toxicities. First, we found that Cu(II), abundant in extracellular regions of human brain (0.5-250 μM), is strongly bound to αSyn (Ka ~108 M-1), leading to the formation of shortened, -sheet enriched αSyn fibrils (<0.2 μm). These Cu(II)-associated fibrils exhibited highly elevated transmissibility and neurotoxicity. αSyn–Cu(II) complex at the molecular level forms a macrochelate that modulates αSyn fibrillation as biphasic processes: conformational rearrangements of αSyn–Cu(II) monomers promote the formation of aggregate seeds while disrupting the single-dimensional seed growth, thereby kinetically controlling αSyn fibrillation. These observations explain the underlying molecular mechanism of Cu(II)-induced structural polymorphism in αSyn fibrils with distinct biological capacity.
Similar to Cu(II), the binding of multiple Ca2+ ions induces the structural transition of Syn monomers to extended conformations, which promotes rapid Syn fibrillation. Additionally, we observed that Ca2+ induced further interfibrillar aggregation, via electrostatic and hydrophobic interactions. Our results from multiple biophysical methods provide detailed information on the structural change of Syn and the formation process of aggregates in the presence of Ca2+. Our investigation on the strcutural transition of Syn by Ca2+ suggests that Ca2+-mediated distinctive aggregation pathway of Syn would provide possible reason for the formation of proteinaceous inclusions (i.e. Lewy bodies) in degenerating neural cells. Overall, our study would be valuable for understanding the influence of two distinct important divalent metal ions, Cu(II) and Ca2+, on the aggregation of Syn during the pathogenesis of α-synucleinopathies. Our study will be valuable for understanding the influence of dysregulated metal homeostasis in the pathology of α-synucleinopathies.
Peptide and Glycan Mass Spectrometry Imaging as Diagnostic Tool in Cancer Research
Abstract
Imaging Mass Spectrometry (IMS) is typically used to determine the distribution of proteins in fresh frozen tissue. Tryptic Peptide and Glycan Imaging has some advantages over imaging of intact proteins. These include peptide level analysis provides the possibility for identification by matching accurate m/z and in situ MS/MS to high quality LC-MS/MS data obtained through digestion of relevant laser dissected tissue. Formalin-fixed paraffin embedded (FFPE) tissue can be analysed after antigen retrieval. A novel method for investigating tissue-specific N-linked glycans was recently developed by our group on formalin-fixed paraffin-embedded (FFPE) murine kidney. We have used those methods to potentially make diagnostic decisions for patients with endometrial and ovarian cancer.
Here we present the latest developments within our group, including up-to-date methods for analysis of FFPE tissue (e.g. tryptic peptide and PNGase F Glycan MALDI-IMS), a method for linking LC-MS/MS data of peptides to MALDI-IMS data using internal calibrants as well as the generation of the first data for a MALDI-IMS patient and disease specific tryptic peptide database and the use of tissue micro arrays. We present that IMS spatially profiles glycoforms in tissue-specific regions, while through liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) the corresponding glycol compositions are structurally characterized. These methods are applied to endometrial and ovarian cancer FFPE tissues.
Metastasis is a crucial step of malignant progression and remains the primary cause of death from solid cancers. In endometrial cancer lymph node metastasis is a crucial factor in the choice of treatment and prognosis of patients. As it is impossible to accurately predict lymphatic metastasis in individual patients, a large number of women who would be cured by local treatment alone, undergo radical surgery including lymph node dissections. Peptide Imaging Mass Spectrometry is used in combination with laser capture dissection and LC-MS/MS to distinguish if patients have metastasis by analysing their primary tumour using FFPE tissue from large patient cohorts. Ovarian cancer is the most fatal gynaecological malignancy in adult women with a five-year overall survival rate of 30%. Patients presented with advanced metastatic disease, requiring tumour debulking surgery and chemotherapy 80% of patients who receive chemotherapy suffer disease relapse, 20% of patients who receive chemotherapy do not respond to treatment at all. We have used MSI and LC-MS/MS on FFPE tissue of chemotherapy responder and non-responders to distinguish them in order to tailor their treatment.
We show the potential of tryptic peptide and glycan imaging on FFPE tissue as a diagnostic tool for metastasis of endometrial cancer and for chemotherapy response in ovarian cancer to give surgeons and oncologists a decision making tool to personalize the treatment of their patients.
Top down proteomics and its application for clinical biomarker
Abstract
State-of-art mass spectrometry along with improvement of both instrumentation and software has accelerated the power of proteomics. Traditionally, bottom up proteomics has played a pivot role in wide spectrum of application including basic biology and biomarker discovery based on quantification and qualification of peptides. And recently, combination of high resolution mass spectrometer and software enable to investigate proteoforms and to characterize post-translational modification (PTM) on intact protein, which have been called as Top down proteomics. Top down proteomics can reveal the uncovered protein information such as differentially existed proteoforms in a biological system. Now, not only identification but also quantification of proteoform is available using table top mass spectrometry with numerial Top down data search software. This platform can work in proteoform biomarker discovery in clinical area, especially. Here, principle of Top down proteomics will be introduced and also its application for biomarker development will be addressed, especially focusing on ApoA1/ApoCIII proteoforms in hypercholesterolemia and biomarker candidates for kidney transplantation rejection.
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