Article
Biochemical Research Methods
Conor Jenkins, Benjamin C. Orsburn
Summary: Recent advances in mass spectrometers and sample preparation methods have led to the rapid development of single cell proteomics. However, there is a lack of tools for assessing data quality. This study presents a lightweight python script and graphical user interface called DIDAR, which can quickly evaluate data quality and reduce analysis time.
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
(2023)
Article
Biochemical Research Methods
Benjamin Furtwangler, Nil Uresin, Khatereh Motamedchaboki, Romain Huguet, Daniel Lopez-Ferrer, Vlad Zabrouskov, Bo T. Porse, Erwin M. Schoof
Summary: Improving global proteome characterization in single-cell proteomics is crucial, and this study demonstrates the effectiveness of real-time search and a new acquisition strategy in enhancing quantitative accuracy and proteome coverage.
MOLECULAR & CELLULAR PROTEOMICS
(2022)
Article
Biochemical Research Methods
Aleksandra A. Petelski, Edward Emmott, Andrew Leduc, R. Gray Huffman, Harrison Specht, David H. Perlman, Nikolai Slavov
Summary: The study introduces the SCoPE2 protocol for single-cell protein analysis, which utilizes isobaric mass tags for labeling and achieves cost-effective quantitative protein analysis.
Article
Biochemical Research Methods
Chris D. McGann, William D. Barshop, Jesse D. Canterbury, Chuwei Lin, Wassim Gabriel, Jingjing Huang, David Bergen, Vlad Zabrouskov, Rafael D. Melani, Mathias Wilhelm, Graeme C. McAlister, Devin K. Schweppe
Summary: Sample multiplexed quantitative proteomics assays have improved instrument efficiency and quantitative accuracy through the development of intelligent data acquisition (IDA) strategies. The implementation of a real-time spectral library searching (RTLS) workflow enables intelligent scan triggering and peak selection in milliseconds, leading to improved quantitation of multiplexed samples. The RTLS method expands the IDA toolbox and allows for more efficient and accurate analysis of molecular phenotypes.
JOURNAL OF PROTEOME RESEARCH
(2023)
Review
Biochemical Research Methods
Weiming Ni, Edward X. X. Han, Matthew Cyr, Sean Mackay, Jing Zhou
Summary: Genetically and phenotypically identical immune cell populations can show great heterogeneity in terms of immune functions and protein secretion profiles. Microfluidic chip-based single-cell highly multiplexed secretome proteomics allows for the study of cellular heterogeneity in immune responses at different levels. Polyfunctional T cells that simultaneously produce multiple proteins per cell have been shown to play a crucial role in effective and lasting cellular immunity. Functional proteomic technology can assess cellular functions comprehensively and uniquely define highly polyfunctional cell subsets, providing insights into functional heterogeneity and helping in the identification of predictive biomarkers and potential correlates for optimized immunotherapeutic product design and personalized immunotherapy development, leading to better clinical outcomes.
Review
Biochemistry & Molecular Biology
Yu-Sheng Wang, Jia Guo
Summary: The ability to quantify a large number of varied transcripts in single cells in their native spatial context is crucial for understanding health and disease, and recent advances in multiplexed FISH methods have enabled comprehensive RNA profiling in individual cells.
FRONTIERS IN MOLECULAR BIOSCIENCES
(2021)
Review
Immunology
Alexander Biederstaedt, Gohar Shahwar Manzar, May Daher
Summary: The advent of cellular immunotherapy and precision genome editing has revolutionized cancer treatment, offering new possibilities but also facing certain limitations. Various engineering strategies are being increasingly utilized to overcome present constraints in building more potent cellular therapeutics.
FRONTIERS IN IMMUNOLOGY
(2022)
Article
Immunology
Yunhao Bai, Bokai Zhu, Xavier Rovira-Clave, Han Chen, Maxim Markovic, Chi Ngai Chan, Tung-Hung Su, David R. McIlwain, Jacob D. Estes, Leeat Keren, Garry P. Nolan, Sizun Jiang
Summary: Multiplex imaging technologies can measure over 40 antibody-labeled parameters in single cells, but lateral spillage of signals in densely packed tissues hinders accurate cell-type annotation. The method REDSEA was devised to correct for this spillage, resulting in improved cell-type classification by reducing contaminating signals from neighboring cells and enhancing the recovery of marker signals in multiplexed images.
FRONTIERS IN IMMUNOLOGY
(2021)
Article
Biochemical Research Methods
Jahangir Sufi, Xiao Qin, Ferran Cardoso Rodriguez, Yong Jia Bu, Petra Vlckova, Maria Ramos Zapatero, Mark Nitz, Christopher J. Tape
Summary: Organoids are biomimetic tissue models consisting of multiple cell types and states, with post-translational modification (PTM) signaling networks controlling cellular phenotypes. A novel multiplexed mass cytometry (MC) protocol, TOBis, enables single-cell comparison of PTMs in organoids across different cell types, providing a new approach to study cell type-specific PTM signaling networks. The protocol takes around 3 days and can generate single-cell data for >40 antibodies from millions of cells in a single MC run.
Article
Cell Biology
Tian-Yi Zhu, Shang-Tong Li, Dan-Dan Liu, Xiajun Zhang, Lianqi Zhou, Rong Zhou, Bing Yang
Summary: This study quantifies protein changes during aging in different individuals using single-worm mass spectrometry, and reveals the heterogeneity of aging by reconstructing the aging trajectory and proteomic landscape of C. elegans.
Article
Oncology
Ho Jung An, Eunkyung An, Shahrooz Rabizadeh, Wei-Li Liao, Jon Burrows, Todd Hembrough, Jin Hyung Kang, Chan Kwon Park, Tae-Jung Kim
Summary: Therapeutic guidance in NSCLC patients with discordant ALK test results remains challenging. SRM quantitative multiplexed proteomics can detect ALK and other predictive proteins, providing additional information for treatment decision-making and predicting clinical outcomes.
Article
Chemistry, Analytical
Gabriela Lomeli, Marc Bosse, Sean C. Bendall, Michael Angelo, Amy E. Herr
Summary: Improvements in single-cell protein analysis are necessary for studying the cell-to-cell variation seen in diseases like cancer. MIBI-TOF is a mass spectrometry imaging technology that allows for highly multiplexed protein detection at the single-cell level.
ANALYTICAL CHEMISTRY
(2021)
Article
Oncology
Emma Rorby, Jorgen Adolfsson, Erik Hultin, Thomas Gustafsson, Kourosh Lotfi, Jorg Cammenga, Jan-Ingvar Jonsson
Summary: Fms-related tyrosine kinase 3 (FLT3) receptor plays an important role in acute myeloid leukemia (AML). The study shows that the combination of PKC412 with daunorubicin has a synergistic effect on inhibiting intracellular signaling proteins, while cytarabine antagonizes this effect. The research also highlights the correlation between FLT3 receptor expression and intracellular cytarabine response in AML patients.
EXPERIMENTAL HEMATOLOGY & ONCOLOGY
(2021)
Review
Oncology
Edwin Roger Parra, Marius Ilie, Ignacio I. Wistuba, Paul Hofman
Summary: Over the past decade, there has been a revolution in cancer treatment with the shift towards targeted molecular therapies and immune-based therapies, particularly immune-checkpoint inhibitors (ICIs). However, not all patients with advanced non-small cell lung cancer (aNSCLC) benefit equally from immunotherapy, and there is a need for additional predictive markers for ICIs efficacy. Multiplex immunostaining, which evaluates multiple molecules in the tumor at the same time, shows promise in optimizing patient selection for ICIs. This review aims to explore the role of multiplex immunostaining in immuno-thoracic oncology and its current advantages and limitations in clinical practice.
BRITISH JOURNAL OF CANCER
(2023)
Article
Biophysics
Yang Wang, Gaolian Xu, Mengdi Guan, Qingyang Zhang, Zhiying Wang, Wenhui Chen, Xiaoxiao Yang, Anbang Qiao, Yubo Fan, Xinxia Cai, Zhou Chen, Lingqian Chang, Bo Wei
Summary: An electrochemical quantitative polymerase chain reaction (E-PCR) platform was developed for multiplexed and quantitative analysis of single-based mutations (SbM) in limited and precious samples, achieving ultra-high sensitivity and specificity.
BIOSENSORS & BIOELECTRONICS
(2022)
Article
Biochemistry & Molecular Biology
Sascha Waidmann, Elena Petutschnig, Wilfried Rozhon, Gergely Molnar, Olga Popova, Karl Mechtler, Claudia Jonak
Summary: Chromatin dynamics play a crucial role in regulating transcriptional programs, which is finely adjusted by chromatin remodelling proteins. DEK3 phosphorylation is found to be essential for chromatin function and cellular stress response, influencing nucleosome occupancy, chromatin accessibility, and gene expression, demonstrating a direct link between GSK3-based signaling and chromatin in facilitating transcriptional responses.
Article
Biochemical Research Methods
Claudia Ctortecka, Gabriela Krssakova, Karel Stejskal, Josef M. Penninger, Sasha Mendjan, Karl Mechtler, Johannes Stadlmann
Summary: The combination of single-cell transcriptomics and proteomics has significantly advanced our understanding of basic biology and disease. A novel data analysis pipeline, using DIA and TMT methods, allows for reproducible comparison of protein expression profiles across hundreds of samples.
MOLECULAR & CELLULAR PROTEOMICS
(2022)
Article
Oncology
Lukas Leiendecker, Tobias Neumann, Pauline S. Jung, Shona M. Cronin, Thomas L. Steinacker, Alexander Schleiffer, Michael Schutzbier, Karl Mechtler, Thibault Kervarrec, Estelle Laurent, Kamel Bachiri, Etienne Coyaud, Rajmohan Murali, Klaus J. Busam, Babak Itzinger-Monshi, Reinhard Kirnbauer, Lorenzo Cerroni, Eduardo Calonje, Arno Rutten, Frank Stubenrauch, Klaus G. Griewank, Thomas Wiesner, Anna C. Obenauf
Summary: This study reveals the presence of human papillomavirus 42 (HPV42) in 96% of digital papillary adenocarcinoma (DPA), a cancer occurring on fingers and toes. It shows that HPV42, previously considered nononcogenic, behaves similarly to oncogenic, high-risk HPVs. Machine learning analysis indicates that HPV-driven transformation induces a germ cell-like transcriptional program conserved across all HPV-driven cancers, with implications for early detection, diagnosis, and therapy.
Article
Biochemical Research Methods
Sebastian Dorl, Stephan Winkler, Karl Mechtler, Viktoria Dorfer
Summary: Spectral library search enables more sensitive peptide identification in tandem mass spectrometry experiments, but suffers from limited availability of high-quality libraries and the difficulty of creating decoy spectra for result validation. MS Ana is a new spectral library search engine that addresses these issues by allowing the use of curated or predicted libraries and providing robust false discovery control through its own decoy library generation algorithm. In benchmark tests, MS Ana outperformed database search, achieving 36% more spectrum matches and 4% more proteins identified in single-shot human cell-line data. The quality of result validation was demonstrated through tests on synthetic peptide pools, and the importance of library selection was highlighted by comparing the performance of different publicly available human spectral libraries.
JOURNAL OF PROTEOME RESEARCH
(2023)
Article
Multidisciplinary Sciences
Friederike Leesch, Laura Lorenzo-Orts, Carina Pribitzer, Irina Grishkovskaya, Josef Roehsner, Anastasia Chugunova, Manuel Matzinger, Elisabeth Roitinger, Katarina Belacic, Susanne Kandolf, Tzi-Yang Lin, Karl Mechtler, Anton Meinhart, David Haselbach, Andrea Pauli
Summary: Using mass spectrometry and cryo-electron microscopy, the study provides molecular evidence that ribosomes transition from a dormant state to an active state during early embryogenesis. Dormant ribosomes are associated with four conserved factors that stabilize ribosomes and repress translation. Addition of recombinant zebrafish Dap1b protein can block translation and reconstitute the dormant ribosome state in vitro.
Article
Chemistry, Analytical
Manuel Matzinger, Elisabeth Mueller, Gerhard Duernberger, Peter Pichler, Karl Mechtler
Summary: We report a comprehensive workflow that includes improved strategies for all steps, from cell lysis to data analysis. The workflow is easy to implement even for novice users, thanks to the use of convenient-to-handle 1 μL sample volume and standardized 384-well plates. Using advanced micro-pillar columns, ultrashort gradient lengths down to 5 minutes were tested for high throughput. By benchmarking different acquisition methods and data analysis algorithms, the workflow achieved identification of 1790 proteins in a single cell using data-dependent acquisition (DDA), and more than 2200 proteins from single-cell level input using data-independent acquisition (DIA) in a 20-minute active gradient. The workflow also demonstrated its suitability for differentiating two cell lines, indicating its potential for cellular heterogeneity determination.
ANALYTICAL CHEMISTRY
(2023)
Article
Biochemistry & Molecular Biology
Leonid Serebreni, Lisa-Marie Pleyer, Vanja Haberle, Oliver Hendy, Anna Vlasova, Vincent Loubiere, Filip Nemcko, Katharina Bergauer, Elisabeth Roitinger, Karl Mechtler, Alexander Stark
Summary: Different classes of promoters have distinct mechanisms of transcription initiation, resulting in either focused or dispersed initiation patterns.
Review
Biochemical Research Methods
Manuel Matzinger, Rupert L. Mayer, Karl Mechtler
Summary: Mapping proteomic fingerprints to transcriptomic data is crucial for understanding gene expression and phenotype. Despite challenges in protein amplification and lack of established gold standard workflows, advances in microfluidics, sample separation, data acquisition, and analysis have improved the analysis of tiny sample amounts. Sensitivity, robustness, and throughput are still urgent needs, and label-free single-cell mass spectrometry is a promising strategy for unbiased quantification. This review focuses on recent advances in label-free single-cell mass spectrometry workflows and provides guidance for method selection and future prospects.
Article
Multidisciplinary Sciences
Andreas Lackner, Michael Mueller, Magdalena Gamperl, Delyana Stoeva, Olivia Langmann, Henrieta Papuchova, Elisabeth Roitinger, Gerhard Duernberger, Richard Imre, Karl Mechtler, Paulina A. A. Latos
Summary: The Erf-NCoR1/2 complex controls trophoblast differentiation by linking signalling with transcriptional repression.
NATURE COMMUNICATIONS
(2023)
Article
Biology
Jaydeep Sidhaye, Philipp Trepte, Natalie Sepke, Maria Novatchkova, Michael Schutzbier, Gerhard Duernberger, Karl Mechtler, Juergen A. Knoblich
Summary: By analyzing the transcriptome and epigenome during human corticogenesis, important gene regulatory networks have been identified. Through the use of human brain organoids, specific transcriptome and proteome analyses were conducted, revealing gene expression modules during cortical development. One module involving mTOR-mediated regulation of translation was investigated, showing that partial inhibition of ribosomal genes translation prevents premature translation of differentiation markers, crucial for maintaining the accuracy of cortical development.
Article
Biochemical Research Methods
Micha J. J. Birklbauer, Manuel Matzinger, Fraenze Mueller, Karl Mechtler, Viktoria Dorfer
Summary: In the paper "Cross-linking Mass Spectrometry 2.0", the authors present an updated version of the MS Annika search engine, which accurately identifies cross-linked peptides and introduces a novel scoring function. The evaluation shows that MS Annika 2.0 outperforms other search engines in terms of true unique cross-link detection and false discovery rate estimation.
JOURNAL OF PROTEOME RESEARCH
(2023)
Article
Plant Sciences
Chao Feng, Elisabeth Roitinger, Otto Hudecz, Maria Cuacos, Jana Lorenz, Veit Schubert, Baicui Wang, Rui Wang, Karl Mechtler, Stefan Heckmann
Summary: During meiotic prophase I, the meiotic chromosome axis is essential for synapsis and meiotic recombination progression. TurboID-based proximity labelling enables the identification of proximate proteins in meiotic cells of A. thaliana. This proteomic profiling helps uncover known and new meiotic proteins in rare cell types like meiotic cells.
Article
Multidisciplinary Sciences
Friederike Leesch, Laura Lorenzo-Orts, Carina Pribitzer, Irina Grishkovskaya, Josef Roehsner, Anastasia Chugunova, Manuel Matzinger, Elisabeth Roitinger, Katarina Belacic, Susanne Kandolf, Tzi-Yang Lin, Karl Mechtler, Anton Meinhart, David Haselbach, Andrea Pauli
Summary: Using mass spectrometry and cryo-electron microscopy analyses, we show that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis in zebrafish and Xenopus laevis. Dormant ribosomes are associated with conserved factors that form two modules, responsible for stabilizing ribosomes and repressing translation. A newly discovered translational inhibitor, Dap1b, is found to play a key role in maintaining the dormant state of ribosomes.
Article
Biochemistry & Molecular Biology
Susanne Kandolf, Irina Grishkovskaya, Katarina Belacic, Derek L. Bolhuis, Sascha Amann, Brent Foster, Richard Imre, Karl Mechtler, Alexander Schleiffer, Hemant D. Tagare, Ellen D. Zhong, Anton Meinhart, Nicholas G. Brown, David Haselbach
Summary: In this study, we determined the structure of the 26S proteasome from Spinacia oleracea, providing the first structural basis for the plant proteasome. We found that the overall architecture of the spinach proteasome is similar to that of proteasomes from mammals and yeast, with a structural difference in the proteolytic active beta 1 subunit. Additionally, we identified a previously unseen compression state in the proteasome's conformational landscape, which may contribute to peptide release after proteolysis. These findings have important implications for further studies on plant proteasomes.
PLANT COMMUNICATIONS
(2022)
