Project description:Analysis of fluorescent assembly B-phycoerythrin with a combination of bottom-up and top-down mass spectrometry to reveal heterogeneity of proteoforms and characterize their chromophorylations.
Project description:Ribosomal purifications of spinach 70S ribosome and human 40S and 60S ribosomal subunits were analyzed with bottom-up LC-MS/MS to identify proteins comprising purified complexes as well as co-purified proteins. In order to assess and quantify proteoforms of ribosomal proteins in ribosomal purifications we employed top-down LC-MS/MS techniques with optimized methods, wherein apart from standard parameters (e.g. collision voltage and dynamic exclusion time) high-resolution and low-resolution were alternately employed in full MS mode.
Project description:Here we present a high-performance software for proteome analysis that combines different mass spectrometric approaches, such as, top-down for intact protein analyses and both bottom-up and middle-down, for proteolytic fragment characterization.
Project description:Selecting a sample preparation strategy for mass spectrometry-based proteomics is critical to the success of quantitative workflows. Here we present a universal, solid-phase protein preparation (USP3) method which is rapid, robust and scalable, facilitating high-throughput protein sample preparation for bottom-up and top-down mass spectrometry (MS) analysis.
Project description:A combination of covalent labelling techniques and mass spectrometry (MS) is currently a progressive approach for the de-riving insights relating to the mapping of protein surfaces or protein-ligand interactions. In this study, we mapped an interac-tion interface between DNA binding domain (DBD) of FOXO4 protein and DNA binding element (DAF16) using the Fast Photochemical Oxidation of Protein (FPOP). Residues involved in protein – DNA interaction were identified using bottom-up approach. To avoid a misinterpretation of obtained data, caused by possible multiple radical oxidation leading to the alter-ing a protein surface and oxidation of deep-buried amino acid residues, a top-down approach was employed for the first time in FPOP analysis. An isolation of singly-oxidized ions allowed their gas-phase separation from multiply oxidized species followed by CID and ECD fragmentation. Application of both fragmentation techniques allowed to obtained complemen-tary fragment sets, out of which the regions shielded in the presence of DNA were deduced. The results obtained by bottom-up and top-down approaches were in a high consistency. Lastly, FPOP results were compared with HDX study of FOXO4-DBD•DAF16 complex. No contradictions were found between both methods. Moreover, their combination provide com-plementary information related to the structure and dynamics of the protein-DNA complex.
Project description:The genus Bothrops is responsible for most part of envenomation accidents in Brazil. Bothrops pubescens is an endemic and neglected species in the Brazilian Pampa Biome. The characterization of its venom is essential since there is no data about it and can be helpful in the discovery of active biomolecules and for a better understanding of its action. We used top-down (TDP), native top-down, and bottom-up proteomic (BUP) approaches to characterize the venom of B. pubescens. We were able to identify 89 protein groups with the BUP approach and 40 unique proteoforms with the TDP approach, demonstrating the similarities and peculiarities of B. pubescens venom. We also identified a dimeric L-amino acid oxidase with using native TDP. Here we present for the first time a bothropic venom characterization through TDP approaches.