pFind Studio: a computational solution for mass spectrometry-based proteomics
2019
JCI Insight2019. Ebrahimi-Nik, H et al.
Univ Connecticut, Sch Med, Dept Immunol, Farmington, CT 06032 USA.
ABSTRACT:Neoepitopes are the only truly tumor-specific antigens. Although potential neoepitopes can be readily identified using genomics, the neoepitopes that mediate tumor rejection constitute a small minority, and there is little consensus on how to identify them. Here, for the first time to our knowledge, we use a combination of genomics, unbiased discovery mass spectrometry (MS) immunopeptidomics, and targeted MS to directly identify neoepitopes that elicit actual tumor rejection in mice. We report that MS-identified neoepitopes are an astonishingly rich source of tumor rejection-mediating neoepitopes (TRMNs). MS has also demonstrated unambiguously the presentation by MHC I, of confirmed tumor rejection neoepitopes that bind weakly to MHC I; this was done using DCs exogenously loaded with long peptides containing the weakly binding neoepitopes. Such weakly MHC I-binding neoepitopes are routinely excluded from analysis, and our demonstration of their presentation, and their activity in tumor rejection, reveals a broader universe of tumor-rejection neoepitopes than presently imagined. Modeling studies show that a mutation in the active neoepitope alters its conformation such that its T cell receptor-facing surface is substantially altered, increasing its exposed hydrophobicity. No such changes are observed in the inactive neoepitope. These results broaden our understanding of antigen presentation and help prioritize neoepitopes for personalized cancer immunotherapy.
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EuPA Open Proteomics2019. Niu, Lili et al.
Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
ABSTRACT:In this study, we faced the challenge of deciphering a protein that has been designed and expressed by E. coli in such a way that the amino acid sequence encodes two concatenated English sentences. The letters 'O' and 'U' in the sentence are both replaced by 'K' in the protein. The sequence cannot be found online and carried to-be-discovered modifications. With limited information in hand, to solve the challenge, we developed a workflow consisting of bottom-up proteomics, de novo sequencing and a bioinformatics pipeline for data processing and searching for frequently appearing words. We assembled a complete first question: "Have you ever wondered what the most fundamental limitations in life are?" and validated the result by sequence database search against a customized FASTA file. We also searched the spectra against an E. coli proteome database and found close to 600 endogenous, co-purified E. coli proteins and contaminants introduced during sample handling, which made the inference of the sentence very challenging. We conclude that E. coli can express English sentences, and that de novo sequencing combined with clever sequence database search strategies is a promising tool for the identification of uncharacterized proteins. 2019 Published by Elsevier B.V. on behalf of European Proteomics Association (EuPA).
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Biochemical Journal2019. Zhang, YY et al.
Chinese Acad Sci, Inst Genet & Dev Biol, State Key Lab Mol Dev Biol, 1 West Beichen Rd, Beijing 100101, Peoples R China.
ABSTRACT:The mitogen-activated protein kinase ERK1/2 (ERKs, extracellular-regulated protein kinases) plays important roles in a wide spectrum of cellular processes and have been implicated in many disease states. The spatiotemporal regulation of ERK activity has been extensively studied. However, scarce information has been available regarding the quality control of the kinases to scavenge malfunctioning ERKs. Using site-specific mutagenesis and mass spectrometry, we found that the disruption of the conserved H-bond between Y210 and E237 of ERK1 through point mutation at or naturally occurring nitration on Y210 initiates a quality control program dependent on chaperon systems and CHIP (C-terminal of Hsp70-interacting protein)-mediated ubiquitination and degradation. The H-bond is also important for the quality control of ERK2, but through a distinct mechanism. These findings clearly demonstrate how malfunctioning ERKs are eliminated when cells are in certain stress conditions or unhealthy states, and could represent a general mechanism for scavenging malfunctioning kinases in stress conditions.
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The Plant Cell2019. Zhao, Ji-Long et al.
1; College of Life Science, Hebei Normal University, Hebei Key Laboratory of 7; Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology 8; of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, 9; Shijiazhuang, Hebei 050024, China
ABSTRACT:The receptor-like kinase SIT1 acts as a sensor in rice (Oryza sativa) roots, relaying salt stress signals via elevated kinase activity to enhance salt sensitivity. Here, we demonstrate that Protein Phosphatase 2A (PP2A) regulatory subunit B'kappa constrains SIT1 activity under salt stress. B'kappa-PP2A deactivates SIT1 directly by dephosphorylating the kinase at Thr515/516, a salt-induced phosphorylation site in the activation loop that is essential for SIT1 activity. B'kappa overexpression suppresses the salt sensitivity of rice plants expressing high levels of SIT1, thereby contributing to salt tolerance. B'kappa functions in a SIT1 kinase-dependent manner. During early salt stress, activated SIT1 phosphorylates B'kappa; this not only enhances its binding with SIT1, it also promotes B'kappa protein accumulation via Ser502 phosphorylation. Consequently, by blocking SIT1 phosphorylation, B'kappa inhibits and fine-tunes SIT1 activity to balance plant growth and stress adaptation. 2019 American Society of Plant Biologists. All rights reserved.
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Talanta2019. Jiang, BY et al.
Fudan Univ, Peoples Hosp Shanghai 5, Shanghai 200433, Peoples R China.
ABSTRACT:N-glycosylation is deeply involved in many biological processes, and approximately 50% of mammalian proteins are predicted to be glycosylated. Many large-scale studies have been carried out to reveal the glycosylation status involved in different physiological pathologies across species. However, the lack of a highly specific and high throughput N-glycosylated enrichment method not only results in extended time requirements but also limits the depth of mapping when handling a large number of samples. In this study, we firstly optimized traditional zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) enrichment and found that using of 70% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) as the enrichment buffer, 2800 g as the washing speed and 600 mu L as the washing volume achieved the best specificity, which is higher than 75%. On this basis, we developed a multi-parallel enrichment strategy assisted by a filter-coated 96-well plate, which achieved high specificity and high throughput simultaneously. This strategy allowed us to enrich large numbers of fractionated samples from hepatocellular carcinoma (HCC) cell lines in less than 2 h. Its good specificity helped us achieve in-depth mapping of the N-glycoproteome in metastatic HCC cell lines. A total of 5466 N-glycosites from 2383 glycoproteins were identified, among which 1900 N-glycosites were unannotated in UniProt. The in-depth glycoproteome mapping provides insight into the N-glycosylation status in HCC cell lines with differences in metastatic potential and contributes to biomarker discovery.
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Journal of Biological Chemistry2019. Roy, Neeladri Sekhar et al.
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT:Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Delta 17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1's activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP(2)), stimulated GAP activity on an N terminus-containing variant, [L8K]ARF1, but only marginally affected activity on [Delta 17]ARF1. A peptide comprising residues 2-17 of ARF1 ([2-17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first beta-strand of the PH domain. Point mutations in either the linker or the C-terminal alpha-helix of the PH domain decreased [2-17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.
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Nature Communications2019. Gowravaram, M et al.
Free Univ Berlin, Inst Chem & Biochem, Takustr 6, D-14195 Berlin, Germany.
ABSTRACT:The mammalian Staufen proteins (Stau1 and Stau2) mediate degradation of mRNA containing complex secondary structures in their 3'-untranslated region (UTR) through a pathway known as Staufen-mediated mRNA decay (SMD). This pathway also involves the RNA helicase UPF1, which is best known for its role in the nonsense-mediated mRNA decay (NMD) pathway. Here we present a biochemical reconstitution of the recruitment and activation of UPF1 in context of the SMD pathway. We demonstrate the involvement of UPF2, a core NMD factor and a known activator of UPF1, in SMD. UPF2 acts as an adaptor between Stau1 and UPF1, stimulates the catalytic activity of UPF1 and plays a central role in the formation of an SMD-competent mRNP. Our study elucidates the molecular mechanisms of SMD and points towards extensive cross-talk between UPF1-mediated mRNA decay pathways in cells.
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Biochemical Journal2019. Friedrich, MG et al.
Univ Wollongong, Illawarra Hlth & Med Res Inst, Wollongong, NSW 2522, Australia.
ABSTRACT:Long-lived proteins (LLPs) are present in numerous tissues within the human body. With age, they deteriorate, often leading to the formation of irreversible modifications such as peptide bond cleavage and covalent cross-linking. Currently understanding of the mechanism of formation of these cross-links is limited. As part of an ongoing study, proteomics was used to characterise sites of novel covalent cross-linking in the human lens. In this process, Lys residues were found cross-linked to C-terminal aspartates that had been present in the original protein as Asn residues. Cross-links were identified in major lens proteins such as alpha A-crystallin, alpha B-crystallin and aquaporin 0. Quantification of the level of an AQP0/AQP0 cross-linked peptide showed increased cross-linking with age and in cataract lenses. Using model peptides, a mechanism of cross-link formation was elucidated that involves spontaneous peptide bond cleavage on the C-terminal side of Asn residues resulting in the formation of a C-terminal succinimide. This succinimide does not form cross-links, but can hydrolyse to a mixture of C-terminal Asn and C-terminal Asp amide peptides. The C-terminal Asp amide is unstable at neutral pH and decomposes to a succinic anhydride. If the side chain of Lys attacks the anhydride, a covalent cross-link will be formed. This multi-step mechanism represents a link between two spontaneous events: peptide bond cleavage at Asn and covalent cross-linking. Since Asn deamidation and cleavage are abundant age-related modifications in LLPs, this finding suggests that such susceptible Asn residues should also be considered as potential sites for spontaneous covalent cross-linking.
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Analytical Chemistry2019. Zhao, LL et al.
Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China.
ABSTRACT:Chemical cross-linking combined with mass spectrometry (CXMS) has emerged as a powerful tool to study protein structure, conformation, and protein-protein interactions (PPIs). Until now, most cross-linked peptides were generated by using commercial cross-linkers, such as DSS, BS3, and DSSO, which react with the primary amino groups of the lysine residues of proteins. However, trypsin, the most commonly used proteolytic enzyme, cannot cleave the C-terminus of a linked lysine, making the obtained cross-linked peptides longer than common peptides and unfavorable for MS identification and data searching. Herein, we propose an in situ sequential digestion strategy using enzymes with distinct cleavage specificity, named as smart cutter, to generate cross-linked peptides with suitable length so that the identification coverage could improve. Through the application of such a strategy to DSS cross-linked E. coli lysates, additional cross-linked sites (1.3-fold increase) obtained in comparison with those obtained by trypsin-trypsin digestion (2879 vs 1255). Among the different digestion combinations, AspN-trypsin performed the best, with 64% (673/1059) of the cross-linked sites complementary to trypsin-trypsin digestion, which is beneficial to ensure the depth for studying protein structure and PPIs. Taking the 60 kDa chaperonin protein as an example, more than twice the cross-linked sites (30 vs 14) were identified to enrich the protein structure information. In addition, compared to the published protein interaction network for E. coli (http://www.bacteriome.org), 91 potential PPIs were discovered with our strategy, of which 65 have not covered by trypsin-trypsin digestion. Therefore, these results illustrate the great significance of smart-cutter-based CXMS for the revelation of protein structure as well as finding new PPIs.
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Cell2019. Xu, M et al.
Southern Med Univ, Nanfang Hosp, Dept Pathol, Guangzhou 510515, Guangdong, Peoples R China.
ABSTRACT:Liver fibrosis is a very common condition seen in millions of patients with various liver diseases, and yet no effective treatments are available owing to poorly characterized molecular pathogenesis. Here, we show that leukocyte cell-derived chemotaxin 2 (LECT2) is a functional ligand of Tie1, a poorly characterized endothelial cell (EC)-specific orphan receptor. Upon binding to Tie1, LECT2 interrupts Tie1/Tie2 heterodimerization, facilitates Tie2/Tie2 homodimerization, activates PPAR signaling, and inhibits the migration and tube formations of EC. In vivo studies showed that LECT2 overexpression inhibits portal angiogenesis, promotes sinusoid capillarization, and worsens fibrosis, whereas these changes were reversed in Lect2-KO mice. Adeno-associated viral vector serotype 9 (AAV9)-LECT2 small hairpin RNA (shRNA) treatment significantly attenuates fibrosis. Upregulation of LECT2 is associated with advanced human liver fibrosis staging. We concluded that targeting LECT2/Tie1 signaling may represent a potential therapeutic target for liver fibrosis, and serum LECT2 level may be a potential biomarker for the screening and diagnosis of liver fibrosis.
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