Article
Biochemistry & Molecular Biology
Clara-L. Sandmann, Jana F. Schulz, Jorge Ruiz-Orera, Marieluise Kirchner, Matthias Ziehm, Eleonora Adami, Maike Marczenke, Annabel Christ, Nina Liebe, Johannes Greiner, Aaron Schoenenberger, Michael B. Muecke, Ning Liang, Robert L. Moritz, Zhi Sun, Eric W. Deutsch, Michael Gotthardt, Jonathan M. Mudge, John R. Prensner, Thomas E. Willnow, Philipp Mertins, Sebastiaan van Heesch, Norbert Hubner
Summary: This study analyzes the evolutionary origins of 7,264 recently cataloged human short open reading frames (sORFs) and finds that most of them are evolutionarily young and emerged de novo. The researchers also identify 221 previously missed sORFs that potentially encode peptides smaller than the smallest annotated human microprotein. By conducting mass-spectrometry-based interactome screens and cellular assays, the study associates some of these sORFs with mRNA splicing, translational regulation, and endocytosis-related functions.
Review
Cell Biology
Alyssa Zi-Xin Leong, Pey Yee Lee, M. Aiman Mohtar, Saiful Effendi Syafruddin, Yuh-Fen Pung, Teck Yew Low
Summary: This review discusses the history, development, and strategies used for identifying, validating, and characterizing sORFs and microproteins. Methods such as ribosome profiling, mass spectrometry-based proteomics, CRISPR/Cas9 screening, and protein-protein interaction studies are employed for detection and functional characterization. The review also highlights the challenges and potential solutions in identifying and validating these novel entities.
JOURNAL OF BIOMEDICAL SCIENCE
(2022)
Article
Multidisciplinary Sciences
Liam Cassidy, Philipp T. Kaulich, Andreas Tholey
Summary: Microproteins and SEPs can undergo various posttranslational modifications like other proteins. However, traditional proteomic methods face challenges in identifying, quantifying and characterizing proteoforms. Top-down proteomics, in contrast, allows for direct identification of intact proteoforms. This article provides an overview of modified microproteins and SEPs, introduces proteoform terminology, and compares the advantages and disadvantages of traditional bottom-up and top-down proteomic workflows. It also discusses future developments in top-down proteomics for proteoform-centric analysis of microproteins and SEPs.
Review
Biochemical Research Methods
Zheng Zhang, Yujie Li, Wenqian Yuan, Zhiwei Wang, Cuihong Wan
Summary: Short open reading frames (sORFs) have the potential to encode proteins, leading to a new research focus in proteomics. Mass spectrometry (MS) is a significant tool for directly detecting the sequence of sORF encoded peptides (SEPs), but the low abundance and short sequences of SEPs present challenges in detection.
Article
Biochemical Research Methods
Philipp T. Kaulich, Liam Cassidy, Juergen Bartel, Ruth A. Schmitz, Andreas Tholey
Summary: The use of multiple proteases in GeLC-MS/MS analysis can improve the identification and sequence coverage of small proteins and SEPs. Combining data from various proteases helped identify more small proteins and SEPs, enhancing their molecular characterization capabilities.
JOURNAL OF PROTEOME RESEARCH
(2021)
Review
Plant Sciences
Yong Feng, Mengyun Jiang, Weichang Yu, Jiannan Zhou
Summary: The increasing recognition of the roles of short/small open reading frames (sORFs) in recent years is attributed to the development and application of the Ribo-Seq technique, which allows for the identification of sORFs in various organisms. However, caution should be exercised when using RPFs to identify sORFs in plants, particularly for polyploidy species, due to the small size and complexity of the plant genome. This study compares different approaches to the identification of plant sORFs, discusses their advantages and disadvantages, and provides a guide for researchers in choosing the appropriate method for plant sORF studies.
FRONTIERS IN PLANT SCIENCE
(2023)
Article
Biotechnology & Applied Microbiology
Niyati Jain, Felix Richter, Ivan Adzhubei, Andrew J. J. Sharp, Bruce D. D. Gelb
Summary: Using a comparative genomics approach, 3,326 high-confidence small protein-coding open reading frames (smORFs) were identified, and it was found that these smORFs are enriched among disease-associated variants. The study highlights the important functional roles of smORF-encoded peptides in human disease.
Article
Microbiology
Matthew G. Durrant, Ami S. Bhatt
Summary: Research reveals that small open reading frames (smORFs) and their encoded microproteins play central roles in microbes, with a vast unexplored space of smORFs within human-associated microbes. The introduction of SmORFinder combines profile hidden Markov models and deep learning models to predict small protein families enriched for Ribo-seq translation signals. Deep learning models are shown to identify Shine-Dalgarno sequences, deprioritize the wobble position in each codon, and group codon synonyms.
CELL HOST & MICROBE
(2021)
Review
Biochemistry & Molecular Biology
I. A. Sedlov, I. A. Fesenko
Summary: Recent studies have shown that short open reading frames (sORFs, <100 codons) can code for peptides or microproteins that play important roles in both prokaryotic and eukaryotic cells. The translation products of sORFs have been found to regulate various processes, such as modulating the activity of the mitochondrial respiratory chain or muscle cells in mammals. However, identifying and analyzing the function of sORF-encoded peptides or microproteins is a complex task that requires special approaches. This review discusses the analysis of the interactome of sORF-encoded microproteins and describes the current methods used for such studies.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
(2023)
Review
Plant Sciences
K. Bharathan Sruthi, Athira Menon, P. Akash, Eppurath Soniya
Summary: Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides without coding potential. Recent research shows that lncRNAs can code for micropeptides, which are generated from small open reading frames (smORFs) and found in short mRNAs and non-coding RNAs. Although micropeptides play critical roles in plant development and stress, only a few functions have been verified so far.
FRONTIERS IN PLANT SCIENCE
(2022)
Article
Biology
Siyuan Zhao, Jun Meng, Jael Sanyanda Wekesa, Yushi Luan
Summary: Recently, it has been discovered that small open reading frames (sORFs) in long noncoding RNA (lncRNA) can encode small peptides, which can help study the mechanisms of growth and development in organisms. This study proposes a novel computational method, called sORFplnc, that is based on class-imbalance learning to identify sORFs with coding potential in plant lncRNA. Experimental results show that sORFplnc outperforms existing computational methods.
COMPUTERS IN BIOLOGY AND MEDICINE
(2023)
Review
Genetics & Heredity
Preeti Madhav Kute, Omar Soukarieh, Hakon Tjeldnes, David-Alexandre Tregouet, Eivind Valen
Summary: Advancements in genomics and molecular biology have revealed the presence of numerous small open reading frames (sORFs) in various types of transcripts, many of which have been found to have physiological functions and are associated with human diseases. These sORFs represent a potential source of therapeutic targets. Unlike protein-coding genes, the function of an sORF is not solely determined by the encoded peptide, but also by the act of translating the sORF itself. Experimental methods such as ribosome profiling and mass spectrometry have been commonly used to identify and characterize sORFs, but determining their function remains challenging.
FRONTIERS IN GENETICS
(2022)
Article
Biochemical Research Methods
Fengyuan Hu, Jia Lu, Louise S. Matheson, Manuel D. Diaz-Munoz, Alexander Saveliev, Jinbo Xu, Martin Turner
Summary: A computational pipeline named ORFLine was developed to identify and classify smORFs, resulting in the discovery of 5744 unique smORFs in mouse B and T lymphocytes datasets. The study further revealed four novel micropeptides that show evidence of secretion and could potentially serve as mediators of immunoregulatory functions.
Review
Biochemical Research Methods
Liam Cassidy, Philipp T. Kaulich, Sandra Maass, Juergen Bartel, Doerte Becher, Andreas Tholey
Summary: This study highlights the developed bottom-up and top-down proteomics approaches for the analysis of SEPs, including sample preparation, mass spectrometry, data interpretation, quantification, and analysis of post-translational modifications. These methods not only cover the analytics of SEPs but also include the low molecular weight proteome, and can be used for the proteome-wide analysis of proteolytic processing events.
Article
Cell Biology
Ying Yang, Damian Gatica, Xu Liu, Runliu Wu, Rui Kang, Daolin Tang, Daniel J. Klionsky
Summary: This study reports the role of uORF-mediated translational control in regulating multiple Atg proteins in yeast and human cells. These findings suggest that uORF-mediated translational control is a widely used mechanism among ATG genes and provide a model for how some ATG genes bypass translational suppression under stress conditions to maintain proper autophagy levels.
Article
Chemistry, Analytical
Philipp T. Kaulich, Liam Cassidy, Konrad Winkels, Andreas Tholey
Summary: In top-down proteomics, pre-fractionation is crucial for high confidence identification of proteoforms and increased proteome coverage. The use of field asymmetric ion mobility spectrometry (FAIMS) has shown to be beneficial, but only external compensation voltage (CV) stepping has been demonstrated. This study investigated the use of internal CV stepping for single-shot TD analysis and optimized MS parameters for different CVs. The optimized multi-CV method significantly increased the number of identified protein groups and proteoforms without increasing measurement time.
ANALYTICAL CHEMISTRY
(2022)
Article
Biochemistry & Molecular Biology
Tomas Koudelka, Konrad Winkels, Patrick Kaleja, Andreas Tholey
Summary: Proteases were found to hydrolyze peptide bonds specifically, modulating the fate and activity of proteins in various biological processes. N- and C-terminomics play crucial roles in identifying protease substrates and their functions. Different strategies and workflows have been developed to increase sensitivity, coverage depth, and throughput in proteomics research.
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH
(2022)
Article
Biochemistry & Molecular Biology
Lukas Seebauer, Yanni Schneider, Alice Drobny, Sonja Ploetz, Tomas Koudelka, Andreas Tholey, Iryna Prots, Beate Winner, Friederike Zunke, Juergen Winkler, Wei Xiang
Summary: In this study, the impact of aSyn on microtubule organization was investigated in cell models and patient-derived neurons. The results suggest that aSyn aggregation interferes with microtubule organization, leading to impaired neuritic phenotypes in Parkinson's disease.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Biochemical Research Methods
Philipp T. Kaulich, Konrad Winkels, Tobias B. Kaulich, Christian Treitz, Liam Cassidy, Andreas Tholey
Summary: Top-down proteomics analyzes intact proteoforms with all modifications and splice variants. MSTopDiff is a software tool that allows the detection of protein modifications without a database search. It is useful for quality control and parameter evaluation in top-down proteomics research.
JOURNAL OF PROTEOME RESEARCH
(2022)
Article
Biochemistry & Molecular Biology
Laura Hobohm, Tomas Koudelka, Fenja H. Bahr, Jule Truberg, Sebastian Kapell, Sarah-Sophie Schacht, Daniel Meisinger, Marion Mengel, Alexander Jochimsen, Anna Hofmann, Lukas Heintz, Andreas Tholey, Matthias Voss
Summary: This study identifies more than 20 substrates of the intramembrane protease SPPL3 and demonstrates its significant role in Golgi enzyme turnover, secretion, and regulation of global glycosylation pathways.
CELLULAR AND MOLECULAR LIFE SCIENCES
(2022)
Article
Cell Biology
Julia Bar, Yannes Popp, Tomas Koudelka, Andreas Tholey, Marina Mikhaylova
Summary: This study reveals that intracellular Ca2+ is necessary for efficient microtubule detyrosination and that calpains can regulate this process. The identification of the calpain-regulated pathway for microtubule detyrosination is of significant importance for basic and clinical research.
JOURNAL OF CELL SCIENCE
(2022)
Article
Biochemistry & Molecular Biology
Ludwig Werny, Antonia Grogro, Kira Bickenbach, Cynthia Bulck, Fred Armbrust, Tomas Koudelka, Kriti Pathak, Franka Scharfenberg, Martin Sammel, Farah Sheikhouny, Andreas Tholey, Stefan Linder, Christoph Becker-Pauly
Summary: This study investigated the interaction between membrane-type-I matrix metalloproteinase (MT1-MMP) and meprin beta, revealing a potential regulatory connection between the two enzymes.
Article
Biochemical Research Methods
Max K. Steinbach, Jan Leipert, Christine Blurton, Matthias Leippe, Andreas Tholey
Summary: This study demonstrates a highly sensitive sample preparation workflow using digital microfluidics and high-resolution mass spectrometry for nanoproteomics research.
JOURNAL OF PROTEOME RESEARCH
(2022)
Article
Multidisciplinary Sciences
Liam Cassidy, Philipp T. Kaulich, Andreas Tholey
Summary: Microproteins and SEPs can undergo various posttranslational modifications like other proteins. However, traditional proteomic methods face challenges in identifying, quantifying and characterizing proteoforms. Top-down proteomics, in contrast, allows for direct identification of intact proteoforms. This article provides an overview of modified microproteins and SEPs, introduces proteoform terminology, and compares the advantages and disadvantages of traditional bottom-up and top-down proteomic workflows. It also discusses future developments in top-down proteomics for proteoform-centric analysis of microproteins and SEPs.
Article
Chemistry, Multidisciplinary
Jan Leipert, Philipp T. Kaulich, Max K. Steinbach, Britta Steer, Konrad Winkels, Christine Blurton, Matthias Leippe, Andreas Tholey
Summary: Researchers have developed a sample preparation method based on protein aggregation and acidic elution, allowing for sensitive protein identification analysis of single nematodes on a digital microfluidics device. Compared to in-tube sample preparation, this method increases proteoform identifications by 46%. Label-free quantification also reveals changes in proteoform abundance that cannot be distinguished by bottom-up proteomics. This workflow facilitates proteoform-focused analysis on limited availability samples.
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
(2023)
Article
Biochemistry & Molecular Biology
Johanna Rueter, Gerald Rimbach, Christian Treitz, Anke Schloesser, Kai Lueersen, Andreas Tholey, Patricia Huebbe
Summary: The study identified protein-protein interactions of APOE with BCKDHA and VDAC1, suggesting a possible involvement of APOE in mitochondrial processes and adaptation to hepatic energy demands. The activity of the BCKD enzyme complex was higher in obese APOE4 mice, while the interaction with VDAC1 was induced upon dietary restriction.
CELLULAR AND MOLECULAR LIFE SCIENCES
(2023)
Article
Biochemical Research Methods
Philipp T. Kaulich, Liam Cassidy, Andreas Tholey
Summary: Efficient proteoform separation is crucial for top-down (TD) proteomics. A two-dimensional low pH/low pH reversed-phase liquid chromatography separation scheme was developed. The optimized separation scheme led to the identification of more than 4900 proteoforms from 1250 protein groups in Caco-2 cells. The low/low pH separation strategy resulted in a higher number of proteoform identifications below 20 kDa compared to the previously published high pH/low pH strategy.
Article
Biochemical Research Methods
Ayako Takemori, Philipp T. Kaulich, Ryo Konno, Yusuke Kawashima, Yuto Hamazaki, Ayuko Hoshino, Andreas Tholey, Nobuaki Takemori
Summary: Middle-down proteomics (MDP) is an analytical method that uses proteases to generate large peptides for mass spectrometry analysis. It is particularly useful for studying high-molecular-weight proteins. In this study, we applied a multidimensional separation workflow combining gel-based prefractionation with LC-FAIMS MS for deep MDP. By improving the digestion conditions and implementing size fractionation and additional ion mobility fractionation, we were able to increase the detectable peptide length and explore proteoform information not covered by conventional top-down proteomics.
Article
Microbiology
Jerome Genth, Kathrin Schaefer, Liam Cassidy, Simon Graspeuntner, Jan Rupp, Andreas Tholey
Summary: Short open reading frames (sORFs) can encode small proteins called sORF-encoded peptides (SEP) that play important roles in various physiological processes. Using proteomics techniques, researchers identified 45 novel and previously reported SEPs in single cultures of Blautia producta under different growth conditions and stress exposure. The results suggest that the biosynthesis of specific SEPs is not limited to bacterial interactions within the microbiome but is influenced by growth and environmental conditions during cultivation.
MICROBIOLOGY SPECTRUM
(2023)
Article
Biochemical Research Methods
Kyowon Jeong, Philipp T. Kaulich, Wonhyeuk Jung, Jihyung Kim, Andreas Tholey, Oliver Kohlbacher
Summary: Top-down proteomics provides more comprehensive proteoform-level information, but reliable data analysis remains challenging. The conventional FDR estimation method may not work at the proteoform level, and the precursor deconvolution error rate should be taken into account.