Identification of cross-linked peptides from complex samples

Yang, Bing; Wu, Yanjie; Zhu, Meiping; Fan, Shidong; Lin, Jinzhong; Zhang, Kun; Li, Shuang; Chi, Hao; Li, Yuxin; Chen, Haifeng; Luo, Shukun; Ding, Yue-He; Wang, Le-Heng; Hao, Zhiqi; Xiu, Li-Yun; Chen, She; Ye, Keqiong; He, Shan; Dong, Meng‐Qiu

doi:10.1038/nmeth.2099

Cited by 533 publications

(631 citation statements)

References 18 publications

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“…This approach enables us to map subunit regions that are in close proximity in the context of the core Atg1 pentameric complex. 33 We first incubated the purified complex with increasing concentrations of either the DSS or BS 3 chemical crosslinker, which covalently conjugates 2 free amine groups over a Ca-Ca distance of approximately 24 to 30 A . 34 Addition of crosslinker led to the appearance of a high molecular mass band above the 250-kDa molecular mass marker, a size that corresponded well to the calculated total mass of the mini pentameric assembly (»350 kDa) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The mass spectrometry data were subsequently analyzed using the pLink program. 33 A total of 163 unique crosslinked peptides were identified, and of these, 74 were "interlinks" or crosslinks between subunits of the Atg1 complex (Table S1). There is a great overlap between the in-gel digestion result and the in-solution digestion result.…”

Section: Resultsmentioning

confidence: 99%

“…One set of TCA precipitates was digested with Lys-C (Promega, V1071) and trypsin (Sigma, T6567) as described before. 33 Another set of TCA precipitates was separated on 5-20% gradient gels (Bio-Rad, 161-1393) followed by Coomassie brilliant blue G-250 staining. The higher molecular weight band corresponding to the crosslinked complex was excised and digested in gel with 5 ng/mL Lys-C and 10 ng/mL trypsin following an established protocol.…”

Section: Electron Microscopymentioning

confidence: 99%

“…CXMS data analysis using pLink Using the pLink search engine, 33 crosslinked peptides were identified by searching the mass spec data against a database containing the protein sequences of Atg1, Atg13, Atg17, Atg31, Atg29, Lys-C and trypsin. The pLink search parameters were: maximum number of missed cleavages (excluding the crosslinking site) D 3; min peptide length D 4 amino acids; fixed modification D cysteine carbamidomethylation.…”

Section: Electron Microscopymentioning

confidence: 99%

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Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Chew¹,

Liu³

et al. 2015

Autophagy

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(2015) Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms, Autophagy, 11:6, 891-905, DOI: 10.1080/15548627.2015 The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1 [CTD]) and Atg13 (Atg13 [CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex. Our data revealed that the intrinsically unstructured C-terminal domain of Atg29 interacts directly with Atg17, whereas Atg17 interacts with Atg13 in 2 distinct intrinsically unstructured regions, including a previously unknown motif that encompasses several putative phosphorylation sites. The Atg1[CTD] crosslinks exclusively to the Atg13[CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13[CTD] and Atg1 [CTD] localize to the tip regions of Atg17-Atg31-Atg29 and do not alter the distinct curvature of this scaffolding subcomplex. This work provides a comprehensive understanding of the subunit interactions in the fully assembled Atg1 core complex, and uncovers the potential role of intrinsically disordered regions in regulating complex integrity.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Electron Microscopymentioning

confidence: 99%

Section: Electron Microscopymentioning

confidence: 99%

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Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Chew¹,

Liu³

et al. 2015

Autophagy

Self Cite

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“…The raw data were searched by pLink 69 using a FASTA database containing 34 NPC protein sequences. An initial MS1 search window of 5 Da was allowed to cover all isotopic peaks of the cross-linked peptides.…”

Section: Methodsmentioning

confidence: 99%

Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

490

894

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Summary Despite the central role of Nuclear Pore Complexes (NPCs) as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm, their large size and dynamic nature have impeded a full structural and functional elucidation. Here, we have determined a subnanometer precision structure for the entire 552-protein yeast NPC by satisfying diverse data including stoichiometry, a cryo-electron tomography map, and chemical cross-links. The structure reveals the NPC’s functional elements in unprecedented detail. The NPC is built of sturdy diagonal columns to which are attached connector cables, imbuing both strength and flexibility, while tying together all other elements of the NPC, including membrane-interacting regions and RNA processing platforms. Inwardly-directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized in distinct functional units. Taken together, this integrative structure allows us to rationalize the architecture, transport mechanism, and evolutionary origins of the NPC.

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DXMSMS Match Program for Automated Analysis of LC‐MS/MS Data Obtained Using Isotopically Coded CID‐Cleavable Cross‐Linking Reagents

Petrotchenko

Makepeace

Borchers

2014

CP in Bioinformatics

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Cross‐linking combined with mass spectrometry for the study of proteins and protein complexes is greatly facilitated by the use of isotopically coded cleavable cross‐linking reagents. The isotopic coding of the cross‐linker enables confident detection of the cross‐link signals, while cleavage of the cross‐linker provides masses of the individual peptides composing the cross‐link and, therefore, facilitates unambiguous assignment of the cross‐links. Here, we describe the DXMSMS Match program, designed for automatic analysis of LC‐MS/MS mass spectrometric data obtained with isotopically coded CID‐cleavable cross‐linkers. The program verifies the assignments of the cross‐links by precursor mass and by inspection of the MS/MS spectra for the fragments and the cleavage products of the cross‐linked peptides. The program produces nonprobabilistic scores for matching the spectra to the theoretical fragmentation of the cross‐links and a visual interface for the validation of the mass spectral matches. © 2014 by John Wiley & Sons, Inc.

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Identification of cross-linked peptides from complex samples

Cited by 533 publications

References 18 publications

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Integrative structure and functional anatomy of a nuclear pore complex

DXMSMS Match Program for Automated Analysis of LC‐MS/MS Data Obtained Using Isotopically Coded CID‐Cleavable Cross‐Linking Reagents

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