LC-MALDI MS/MS Massenspektrometer
Final Report Abstract
This LC-MALDI-MS/MS system has been acquired upon the initiative of 10 research groups of the TUM faculty Wissenschaftszentrum Weihenstephan. Its user base has since expanded considerably and currently encompasses13 research laboratories on campus and is additionally used together with partners across other TUM faculties. Despite the many users, we have not set up a classical core facility to run this instrument. Instead, we have implemented an open access model in which the Kuster group houses and maintains the equipment and trains new users that then utilize the equipment independently in their research projects. Given its large use base, only a select few studies can be highlighted from the past three years but which cover a broad range of scientific scope ranging from analytical method development to the plant scienes and the medical sciences: 1) Phosphorylation site localization in peptides by MALDI MS/MS and the Mascot Delta Score: Using a library of 180 phosphorylated peptides with precisely known modification sites, we (in collaboration with the equipment vendors Waters and Thermo) developed a scoring system that allows the false localisation rate (FLR) determination of peptide phosphorylation in phosphoproteomics studies using MALDI MS/MS. This approach has been adapted by many laboratories around the world, has inspired the development of additional such tools by members of the community and is now implemented in the commonly used commercial protein identification software Mascot so that any laboratory can enjoy the benefit of the developed algorithm. 2) Carbonyl-reactive tandem mass tags for the proteome-wide quantification of N-linked glycans: Tandem mass tags are highly successfully used in proteomic research. However, the existing reagents can only be used to tag peptides for quantitative measurements. In collaboration with ThermoFisher Scientific, we have extended the concept of isobaric tagging to N-linked oligosaccharides released from glycoproteins using TMT derivatives with a carbonyl-reactive functionality. This allowed the quantitative comparison of N-glycan profiles of human cancer cells including isogenic lines with different metastatic potential. In the future, the observed differences in the glycan profiles of the cell lines may be used as surrogates for the assessment of the metastatic potential of tumors from patients. 3) Lactocepin secreted by Lactobacillus exerts anti-inflammatory effects by selectively degrading proinflammatory chemokines: The intestinal microbiota has been linked to inflammatory bowel diseases (IBD), and oral treatment with specific bacteria can ameliorate IBD. One bacterial mixture, VSL#3, containing Lactobacillus, Bifidobacterium, and Streptococcus, was clinically shown to reduce inflammation in IBD patients and normalize intestinal levels of IP-10, a lymphocyte-recruiting chemokine, in a murine colitis model. We identified Lactobacillus paracasei prtP-encoded lactocepin as a protease that selectively degrades secreted, cell-associated, and tissue-distributed IP-10, resulting in significantly reduced lymphocyte recruitment after intraperitoneal injection in an ileitis model. A human Lactobacillus casei isolate was also found to encode lactocepin and degrade IP-10. L. casei feeding studies in a murine colitis model (T cell transferred Rag2(-/-) mice) revealed that a prtP-disruption mutant was significantly less potent in reducing IP-10 levels, T cell infiltration and inflammation in cecal tissue compared to the isogenic wild-type strain. Thus, lactocepin-based therapies may be effective treatments for chemokine-mediated diseases like IBD. 4) Comprehensive proteome analysis in Cenococcum geophilum Fr. as a tool to discover drought-related proteins: Cenococcum geophilum is a widely distributed ectomycorrhizal fungus potentially playing a significant role in resistance and resilience mechanisms of its tree hosts exposed to drought stress. In this study, we performed a large scale protein analysis in pure cultures of C. geophilum in order to gain first global insights into the proteome assembly of this fungus. Using 1-D gel electrophoresis coupled with ESI- MS/MS, we indentified 638 unique proteins. Most of these proteins were related to the metabolic and cellular processes, and the transport machinery of cells. In a second step, we examined the influence of water deprivation on the proteome of C. geophilum pure cultures at three time points of gradually imposed drought. The results indicated that 12 proteins were differentially abundant in mycelia subjected to drought compared to controls. The induced responses in C. geophilum point towards regulation of osmotic stress, maintainance of cell integrity, and counteracting increased levels of reactive oxygen species formed during water deprivation. 5) MALDI imaging mass spectrometry reveals COX7A2, TAGLN2 and S100-A10 as novel prognostic markers in Barrett's adenocarcinoma: To characterize proteomic changes found in Barrett's adenocarcinoma and its premalignant stages, the proteomic profiles of histologically defined precursor and invasive carcinoma lesions were analyzed by MALDI imaging MS. For a primary proteomic screening, a discovery cohort of 38 fresh frozen Barrett's adenocarcinoma patient tissue samples was used. The goal was to find proteins that might be used as markers for monitoring cancer development as well as for predicting regional lymph node metastasis and disease outcome. Using mass spectrometry for protein identification and validating the results by immunohistochemistry on an independent validation set, we could identify two of 60 differentially expressed m/z species between Barrett's adenocarcinoma and the precursor lesion: COX7A2 and S100-A10. Furthermore, among 22m/z species that are differentially expressed in Barrett's adenocarcinoma cases with and without regional lymph node metastasis, one was identified as TAGLN2. In the validation set, we found a correlation of the expression levels of COX7A2 and TAGLN2 with a poor prognosis while S100-A10 was confirmed by multivariate analysis as a novel independent prognostic factor in Barrett's adenocarcinoma. Our results underscore the high potential of MALDI imaging for revealing new biologically significant molecular details from cancer tissues which might have potential for clinical application. This article is part of a Special Issue entitled: Translational Proteomics.
