Project description:We investigated the combined sensitivity of micro-flow liquid chromatography with a ZenoTOF mass spectrometer for high throughput proteomic and phosphoproteomic analysis of rat tissues. Comparing the proteomes acquired using data-independent acquisition (DIA) on the ZenoTOF 7600 with the previous generation TripleTOF 6600, more proteins were quantified using a fifth of the sample load and a third of the instrument time. Zeno SWATH data was evaluated using replicate injections of rat organ digests to compare FragPipe and DIA-NN computational pipelines. FragPipe identified more proteins in 7 of the 8 rat organs, with an extra 12% and 17% observed in heart and muscle tissue respectively. The number of identified peptides per protein were higher with FragPipe and the precision of missing values across replicate injections was more consistent. Single-shot phosphopeptide enrichment from 100 µg rat tissue, without fractionation, was acquired using data-dependent acquisition (DDA) on both instruments. A total of 5,108 phosphosites were quantified with a negligible increase in phosphosites found using the ZenoTOF 7600 relative to the 6600. Using DIA on the ZenoTOF, 8,013 phosphosites were quantified using Spectronaut.
Project description:The retina plays a crucial role in processing and decoding visual information, both in normal development and during myopia progression. Recent advancements have introduced a library-independent approach for data-independent acquisition (DIA) analyses. This study demonstrates deep proteome identification and quantification in individual mice retinas during myopia development, with an average of 6,263 ± 86 unique protein groups. We anticipate that this robust quantification and in-depth retinal-specific spectral library will contribute to a better understanding of the proteome complexity of retina. Furthermore, a comprehensive mice retinal-specific spectral library was generated, encompassing a total identification of 9,401 protein groups, 70,041 peptides, 95,339 precursors, and 761,868 transitions acquired using SWATH-MS acquisition on a ZenoTOF 7600 mass spectrometer. This dataset surpasses the spectral library generated through high-pH reversed-phase fractionation by data-dependent acquisition (DDA). The data is available via ProteomeXchange with the identifier PXD046983. It will also serve as an indispensable reference for investigations in myopia research and other retinal or neurological diseases.
Project description:Example dataset for Annotator tool. Data includes:
- the top-down data of Fab of Herceptin, summed and deconvoluted, measured on Eclipse;
- the bottom-up data of O-glycosylation of human colostrum IgA, measured on ZenoTOF 7600 using EAD;
- the bottom-up data of N-glycosylation of human plasma, measured on ZenoTOF 7600 using EACID.
Project description:Generation of a new library of targeted mass spectrometry assays for accurate protein quantification in triple negative breast cancer (TNBC) tissues. Primary tumor tissue lysates from 105 TNBC patients treated at Masaryk Memorial Cancer Institute (MMCI) in Brno, Czech Republic, were used to generate the spectral library. This project covers raw files from data-dependent acquisition (DDA) – parallel accumulation-serial fragmentation (PASEF) measurements of 12 hydrophilic chromatography (HILIC) fractions of aliquot pool from complete set of 105 samples measured on timsTOF Pro; raw files of 16 individual samples measured in data-independent acquisition (DIA) – PASEF mode and used for hybrid library generation and for demonstrative quantitative DIA data extraction; Pulsar archive generated in Spectronaut 16.0 from 12 DDA-PASEF measurements of HILIC fractions and from 16 data-independent acquisition DIA-PASEF measurements of individual samples. The 16 DIA-PASEF runs of individual samples used for library generation were analyzed using newest versions of Spectronaut (version 18.5) and DIA-NN (version 1.8.1) software tools in library-based setting using the newly generated library as well as in library-free setting showing library-based method to outperform the use of predicted libraries in the terms of identification numbers.
Project description:Protein glycosylation is a critical PTM for the stability and biological function of many proteins, but full characterisation of site-specific glycosylation of proteins remains analytically challenging. Collision induced dissociation (CID) is the most common fragmentation method used in LC-MS/MS workflows, but loss of labile modifications render CID inappropriate for detailed characterisation of site-specific glycosylation. Electron-based dissociation (ExD) methods provide alternatives that retain intact glycopeptide fragments for unambiguous site localisation, but these methods often underperform CID due to increased reaction times and reduced efficiency. Electron activated dissociation (EAD) is another strategy for glycopeptide fragmentation. Here, we use a ZenoTOF 7600 SCIEX instrument to compare the performance of various fragmentation techniques for the analysis of a complex mixture of mammalian O- and N-glycopeptides.
Project description:Proteomic and lipidomic analysis of Ground Demonstrator Model of ArtEMISS-C (Science Verification Test), MELiSSA Project on Limnospira indica PCC 8005 P3.
Proteomic analysis has been performed with AB Sciex TripleTOF 6600.
Lipidomic analysis has been performed with AB Sciex ZenoTOF 7600.
Project description:The study investigated presentation of HLA A2 restricted H3.3K27M neopeptide using immunopeptidomics followed by DDA and/or targeted multiple monitoring reaction (MRM).
Project description:Class-switching to IgG2a/c in mice is a hallmark response to intracellular pathogens. T cells can promote class-switching and the predominant pathway for induction of IgG2a/c antibody responses has been suggested to be via stimulation from Th1 cells. We previously formulated CAF®01 (cationic liposomes containing dimethyldioctadecylammonium bromide (DDA) and Trehalose-6,6-dibehenate (TDB)) with the lipidated TLR7/8 agonist 3M-052 (DDA/TDB/3M-052), which promoted robust Th1 immunity in newborn mice. When testing this adjuvant in adult mice using the recombinant Chlamydia trachomatis (C.t.) vaccine antigen CTH522, it similarly enhanced IgG2a/c responses compared to DDA/TDB, but surprisingly reduced the magnitude of the IFN-g+ Th1 response in a TLR7 agonist dose-dependent manner. Single cell RNA-sequencing revealed that DDA/TDB/3M-052 liposomes initiated early transcription of class-switch regulating genes directly in pre-germinal center B cells. Mixed bone marrow chimeras further demonstrated that this adjuvant did not require Th1 cells for IgG2a/c switching, but rather facilitated TLR7-dependent T-bet programming directly in B cells. This study underlines that adjuvant-directed IgG2a/c class-switching in vivo can occur in the absence of T cell help, via direct activation of TLR7 on B cells and positions DDA/TDB/3M-052 as a powerful adjuvant capable of eliciting type I-like immunity in B cells without strong induction of Th1 responses.