pFind Studio: a computational solution for mass spectrometry-based proteomics
2023
Biophysics Reports2023. Jian-Hua, Wang et al.
National Institute of Biological Sciences (NIBS), Beijing 102206, China
ABSTRACT:Transient and weak protein-protein interactions are essential to many biochemical reactions, yet are technically challenging to study. Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) provides a powerful tool in the analysis of such interactions. Central to this technology are chemical cross-linkers. Here, using two transient heterodimeric complexes EIN/HPr and EIIAGlc/EIIBGlc as our model systems, we evaluated the effects of two amine-specific homo-bifunctional cross-linkers with different reactivities. We showed previously that DOPA2 (di-ortho-phthalaldehyde with a di-ethylene glycol spacer arm) cross-links proteins 60-120 times faster than DSS (disuccinimidyl suberate). We found that though most of the intermolecular cross-links of either cross-linker are consistent with the encounter complexes (ECs), an ensemble of short-lived binding intermediates, more DOPA2 intermolecular cross-links could be assigned to the stereospecific complex (SC), the final lowest-energy conformational state for the two interacting proteins. Our finding suggests that faster cross-linking captures the SC more effectively and cross-linkers of different reactivities potentially probe protein-protein interaction dynamics across multiple timescales.
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Redox Biology2023. Wu, Ting et al.
Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
ABSTRACT:Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on the identification of cross-linked peptides supported by high-quality MS/MS spectra, which eliminates false-positive discoveries of indirect binders. Additionally, cross-linking sites allow the analysis of interaction interfaces, providing further information for substrate validation. We demonstrated this strategy by identifying direct substrates of thioredoxin in both E. coli and HEK293T cells using two bis-vinyl sulfone chemical cross-linkers BVSB and PDES. We confirmed that BVSB and PDES have high specificity in cross-linking the active site of thioredoxin with its substrates both in vitro and in live cells. Applying live cell cross-linking, we identified 212 putative substrates of thioredoxin in E. coli and 299 putative S-nitrosylation (SNO) substrates of thioredoxin in HEK293T cells. In addition to thioredoxin, we have shown that this strategy can be applied to other proteins in the thioredoxin superfamily. Based on these results, we believe future development of cross-linking techniques will further advance cross-linking mass spectrometry in identifying substrates of other classes of enzymes.
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Angewandte Chemie International Edition2023. Liu, Jialin et al.
College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular Sciences, Peking-Tsinghua Center for Life Sciences, Synthetic and Functional Biomolecules Center, and Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, 100871 China
ABSTRACT:
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Proceedings of the National Academy of Sciences2023. de Kock, Willemien et al.
Groningen Institute of Archaeology, Faculty of Arts, University of Groningen, 9712 ER Groningen, Netherlands
ABSTRACT:
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Biochemistry2023. liuhaijun et al.
Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130, United States
ABSTRACT:Phycobilisomes (PBSs) are the major photosynthetic light-harvesting complexes in cyanobacteria and red algae. PBS, a multisubunit protein complex, has two major interfaces that comprise intrinsically disordered regions (IDRs): rod-core and core-membrane. IDRs do not form regular, three-dimensional structures on their own. Their presence in the photosynthetic pigment-protein complexes portends their structural and functional importance. A recent model suggests that PB-loop, an IDR located on the PBS subunit ApcE and C-terminal extension (CTE) of the PBS subunit ApcG, forms a structural protrusion on the PBS core-membrane side, facing the thylakoid membrane. Here, the structural synergy between the rod-core region and the core-membrane region was investigated using quantitative mass spectrometry (MS). The AlphaFold-predicted CpcG-CTE structure was first modeled onto the PBS rod-core region, guided and justified by the isotopically encoded structural MS data. Quantitative cross-linking MS analysis revealed that the structural proximity of the PB-loop in ApcE and ApcG-CTE is significantly disturbed in the absence of six PBS rods, which are attached to PBS via CpcG-CTE, indicative of drastic conformational changes and decreased structural integrity. These results suggest that CpcG-rod attachment on the PBS rod-core side is essentially required for the PBS core-membrane structural assembly. The hypothesized long-range synergy between the rod-core interface (where the orange carotenoid protein also functions) and the terminal energy emitter of PBS must have important regulatory roles in PBS core assembly, light-harvesting, and excitation energy transmission. These data also lend strategies that genetic truncation of the light-harvesting antennas aimed for improved photosynthetic productivity must rely on an in-depth understanding of their global structural integrity.
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Journal of Proteomics2023. Jess Vzquez et al.
Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid 28029, Spain
ABSTRACT:Open-search methods allow unbiased, high-throughput identification of post-translational modifications in proteins at an unprecedented scale. The performance of current open-search algorithms is diminished by experimental errors in the determination of the precursor peptide mass. In this work we propose a semisupervised open search approach, called ReCom, that minimizes this effect by taking advantage of a priori known information from a reference database, such as Unimod or a database provided by the user. We present a proof-of-concept study using Comet-ReCom, an improved version of Comet-PTM. Comet-ReCom increased identification performance of Comet-PTM by 68%. This increased performance of Comet-ReCom to score the MS/ MS spectrum comes in parallel with a significantly better assignation of the monoisotopic peak of the precursor peptide in the MS spectrum, even in cases of peptide coelution. Our data demonstrate that open searches using ultra-tolerant mass windows can benefit from using a semi-supervised approach that takes advantage from previous knowledge on the nature of protein modifications. Significance: The present study introduces a novel approach to ultra-tolerant database search, which employs prior knowledge of post-translational modifications (PTMs) to improve identification of modified peptides. This method addresses the limitations related to experimental errors and precursor mass assignation of previous opensearch methods. Thus, it enables the study of the biological significance of a wider variety of PTMs, including unknown or unexpected modifications that may have gone unnoticed using non-supervised search methods.
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Journal of Proteome Research2023. Kweon, Hye Kyong et al.
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1085, United States
ABSTRACT:Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search. This approach, supported by manual validation, allows the confident identification of sulfotyrosine-containing peptides in the presence of high levels of phosphorylated peptides, thus enabling these two sterically and ionically similar isobaric PTMs to be distinguished and annotated in a single proteomic analysis. We applied this approach to isolated interphase and mitotic rat liver Golgi membranes and identified 67 tyrosine sulfopeptides, corresponding to 26 different proteins. This work discovered 23 new sulfoproteins with functions related to, for example, Ca2+-binding, glycan biosynthesis, and exocytosis. In addition, we report the first preliminary evidence for crosstalk between sulfation and phosphorylation in the Golgi, with implications for functional control.
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2023. Lai, Shengzhi et al.
Department of Electronic and Computer Engineering
ABSTRACT:
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Analytical Chemistry2023. Juho Son et al.
Hanyang Univ, Dept Comp Sci, Seoul 04763, South Korea; Hanyang Univ, Inst Artificial Intelligence Res, Seoul 04763, South Korea
ABSTRACT:Predicting peptide detectability is useful in a varietyof massspectrometry (MS)-based proteomics applications, particularly targetedproteomics. However, most machine learning-based computational methodshave relied solely on information from the peptide itself, such asits amino acid sequences or physicochemical properties, despite thefact that peptides detected by MS are dependent on many factors, includingprotein sample preparation, digestion, separation, ionization, andprecursor selection during MS experiments. DbyDeep (Detectabilityby Deep learning) is an innovative end-to-end LSTM network model forpeptide detectability prediction that incorporates sequence contextsof peptides and their cleavage sites (by protease). Utilizing thecleavage site contexts could improve the performance of prediction,and DbyDeep outperformed existing methods in predicting peptides recognizablefrom multiple MS/MS data sets with diverse species and MS instruments.We argue for the necessity of a learning model that encompasses severalcontexts associated with peptide detection, as opposed to dependingjust on peptide sequences. There is a Python implementation of DbyDeepat https://github.com/BISCodeRepo/DbyDeep.
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Journal of Assisted Reproduction and Genetics2023. Yuqin Yang et al.
Department of Reproductive Medicine, Womens Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, China
ABSTRACT:
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