Mapping Protein Carboxymethylation Sites Provides Insights into their Role in Proteostasis and Cell Proliferation
Di Sanzo S. et al., Nature Communications 2021
Advanced glycation end products (AGEs) result from non-enzymatic glycation of proteins, altering their structure and function. N(6)-carboxymethyllysine (CML) is one of the most abundant AGEs and is known to accumulate in human tissues with age, hence classifying as a biomarker for aging. To characterize CML-targeted proteins and pathways, Di Sanzo and coworkers developed a workflow to enrich CML-modified peptides and employed both SpectroMine and Spectronaut for carboxymethylation site discovery, followed by SpectroDive for biomarker validation. For more information on this study see our SpectroDive launch seminar here.
MhcVizPipe: A Quality Control Software for Rapid Assessment of Small- to Large-Scale Immunopeptidome Data Sets
Kovalchik K.A. et al., MCP 2021
Mass spectrometry (MS)-based immunopeptidomics is a rapidly evolving, automated, and high-throughput technology that produces small- to large-scale datasets of clinically relevant MHC class I- and II-associated peptides. This technology requires the development of software tools for the rapid and simultaneous quality control of large clinical immunopeptidomic studies. Biognosys’ researchers joined an international collaborative effort to develop MhcVizPipe, a semi-automated, fast post-acquisition tool for the assessment of sample quality and MHC-specificity through powerful data visualization and reporting.
Reproducible Determination of High-Density Lipoprotein Proteotypes
Goetze S. et al., Journal of Proteome Research 2021
High-density lipoprotein (HDL) particles are multimolecular complexes known for their beneficial role in maintaining cholesterol homeostasis by transporting cholesterol from peripheral tissues back to the liver. The HDL proteome is complex and heterogeneous, and different proteoforms can affect various biological processes, such as lipid metabolism and acute inflammation. Goetze and colleagues performed robust HDL proteotyping across patient cohorts using a 296 protein group spectral library for DIA analysis in Spectronaut.
DIA-Based Proteome Profiling of Nasopharyngeal Swabs from COVID-19 Patients
Mun D. et al., Journal of Proteome Research 2021
In this study, the Biognosys’ R&D team, in collaboration with researchers of the University of Wisconsin-Madison, co-developed and benchmarked a DIA method to quantify acetylation stoichiometry in the cell proteome. This improved DIA method overcomes the limitations of DDA, allowing the accurate and reproducible quantification of light and heavy acetyl-lysine fragment ions. The method was benchmarked on synchronized cell lines with defined acetylation stoichiometry, providing a detailed landscape of the site-specific acetylation dynamics across cellular compartments.
Molecular Characterization of Hematopoietic Stem Cells after in Vitro Amplification on Biomimetic 3D PDMS Cell Culture Scaffolds
Marx-Blümel L. et al., Scientific Reports 2021
The efficacy of hematopoietic stem cell (HSC) transplantation can be impaired by insufficient numbers of donor cells, and their proliferation in ex-vivo culture systems results in a loss of pluripotency. Marx-Blümel and colleagues developed a 3D polydimethylsiloxane (PDMS) ex vivo cell culture system to grow HSCs and prevent their differentiation. Proteomic characterization of the cell cultures in DIA using Spectronaut’s hybrid library approach, evidenced distinct proteomic signatures and highlighted key signaling pathways in the proliferation and maintenance of pluripotent HSCs.
Unravelling the Ubiquitination of Circadian Clock Proteins with Spectronaut
Fynn M. Hansen F.M. et al., Nature Communications 2021
Ubiquitination plays a pivotal role in the regulation of the circadian clock in mammals. Comprehensive profiling of endogenous ubiquitination is challenging due to its low stoichiometry and varying ubiquitin-chain topologies. Here the authors developed a sensitive workflow that combines diGly antibody-based enrichment, optimized DIA acquisition, and analysis in Spectronaut with spectral libraries that contain over 90,000 diGly peptides. This workflow enabled in-depth diGly proteome coverage in single-shot experiments and uncovered hundreds of cycling ubiquitination sites across the circadian cycle.
Deep Characterization of the HLA Immunopeptidome with Spectronaut
Pak S. et al., MCP 2021
T cells recognize short peptide antigens of 8–11 amino acids in length presented by human leukocyte antigen (HLA) molecules on tumor cells. Deep characterization of the HLA immunopeptidome can advance the development of therapeutics against cancer. Due to the limited amount of HLA peptides eluted from clinical samples, a comprehensive multi-HLA library was generated for DIA analysis in Spectronaut. The use of a complex multi-HLA spectral library, which included sample-specific libraries and libraries that combined up to 40 different immunopeptidomics samples, boosted sensitivity without compromising specificity.
Potential Targets to Treat Drug-resistant Tumors Uncovered with Spectronaut’s directDIA Phosphoproteomics Workflow
Franciosa G. et al., Nature Communications 2021
Deregulation of the Notch signaling pathway has wide-ranging effects across multiple cancer types. The clinical efficacy of γ-secretase inhibitors (GSIs), small molecules that block Notch cleavage and subsequent activation, is limited due to the emergence of drug-resistant tumors. This work systematically compares GSI resistant and sensitive tumor cells by DIA phosphoproteomics, using Spectronaut’s directDIA workflow. The results highlighted a rewiring of the signaling networks upon Notch inhibition and identified druggable pathways in cancer that can be targeted in combination treatments to overcome resistance.
Investigating Age-dependent Changes in Muscle Stem Cell Functionality with Spectronaut and SpectroDive
Schüler S.C. et al., Cell Reports 2021
Muscle stem cells (MuSCs) confer a remarkable regenerative capacity to skeletal muscles, which however, declines during aging due to a reduction in MuSCs number and functionality. Since the interaction of MuSCs with the stem cell niche is critical to provide the proper cues for self-renewal, researchers investigated how aging alters the communication between MuSCs and their niche. Deep proteome DIA analysis in Spectronaut followed by PRM in SpectroDive unraveled aging-affected signaling axes and showed that the accumulation of the secreted protein Smoc2 contributes to their perturbation in MuSCs during aging.
Multi-omics Investigation of Daily Exercise Performance with SpectroMine
Maier G. et al., The Journal of Physiology 2021
Muscle activity has been proposed to be regulated by the circadian clock and to act as an upstream Zeitgeber to synchronize physiological and molecular responses in muscles and other peripheral tissues. Here the authors employed a multi-omic approach to study the circadian regulation of endurance performance and control of the circadian clock by exercise. The quantitative analysis of both the phospho- and total proteome labeled with isobaric TMT tags was conducted in SpectroMine. This study provides a starting point to identify and validate markers of exercise performance, possibly contributing to personalized training design and patient care.
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