Dataset Information

Characterization of In Vivo Protein Complexes via Chemical Cross-Linking and Mass Spectrometry.

ABSTRACT: Cells perform various functions by proteins via protein complexes. Characterization of protein complexes is critical to understanding their biological and clinical significance and has been one of the major efforts of functional proteomics. To date, most protein complexes are characterized by the in vitro system from protein extracts after the cells or tissues are lysed, and it has been challenging to determine which of these protein complexes are formed in intact cells. Herein, we report an approach to preserve protein complexes using in vivo cross-linking, followed by size exclusion chromatography and data-independent acquisition mass spectrometry. This approach enables the characterization of in vivo protein complexes from cells or tissues, which allows the determination of protein complexes in clinical research. More importantly, the described approach can identify protein complexes that are not detected by the in vitro system, which provide unique protein function information.

SUBMITTER: Wang Y

PROVIDER: S-EPMC9006583 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Publications

Characterization of In Vivo Protein Complexes via Chemical Cross-Linking and Mass Spectrometry.

Wang Yuefan Y Hu Yingwei Y Höti Naseruddin N Huang Lan L Zhang Hui H

Analytical chemistry 20211228 3

Cells perform various functions by proteins via protein complexes. Characterization of protein complexes is critical to understanding their biological and clinical significance and has been one of the major efforts of functional proteomics. To date, most protein complexes are characterized by the in vitro system from protein extracts after the cells or tissues are lysed, and it has been challenging to determine which of these protein complexes are formed in intact cells. Herein, we ...[more]

PMID: 34962381

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Project description:BackgroundChemical cross-linking is used for protein-protein contacts mapping and for structural analysis. One of the difficulties in cross-linking studies is the analysis of mass-spectrometry data and the assignment of the site of cross-link incorporation. The difficulties are due to higher charges of fragment ions, and to the overall low-abundance of cross-link species in the background of linear peptides. Cross-linkers non-specific at one end, such as photo-inducible diazirines, may complicate the analysis further. In this report, we design and validate a novel cross-linked peptide mapping algorithm (XLPM) and compare it to StavroX, which is currently one of the best algorithms in this class.ResultsWe have designed a novel cross-link search algorithm -XLPM - and implemented it both as an online tool and as a downloadable archive of scripts. We designed a filter based on an observation that observation of a b-ion implies observation of a complimentary y-ion with high probability (b-y filter). We validated the b-y filter on the set of linear peptides from NIST library, and demonstrate that it is an effective way to find high-quality mass spectra. Next, we generated cross-linked data from an ssDNA binding protein, Rim1with a specific cross-linker disuccinimidyl suberate, and a semi-specific cross-linker NHS-Diazirine, followed by analysis of the cross-linked products by nanoLC-LTQ-Orbitrap mass spectrometry. The cross-linked data were searched by XLPM and StavroX and the performance of the two algorithms was compared. The cross-links were mapped to the X-ray structure of Rim1 tetramer. Analysis of the mixture of NHS-Diazirine cross-linked ¹?N and ¹?N-labeled Rim1 tetramers yielded ¹?N-labeled to ¹?N-labeled cross-linked peptide pairs, corresponding to C-terminus-to-N-terminus cross-linking, demonstrating interaction between different two Rim1 tetramers. Both XLPM and StavroX were successful in identification of this interaction, with XLPM leading to a better annotation of higher-charged fragments. We also put forward a new method of estimating specificity and sensitivity of identification of a cross-linked residue in the case of a non-specific cross-linker.ConclusionsThe novel cross-link mapping algorithm, XLPM, considerably improves the speed and accuracy of the analysis compared to other methods. The quality selection filter based on b-to-y ions ratio proved to be an effective way to select high quality cross-linked spectra.

Dataset Information

Characterization of In Vivo Protein Complexes via Chemical Cross-Linking and Mass Spectrometry.

Publications

Characterization of <i>In Vivo</i> Protein Complexes <i>via</i> Chemical Cross-Linking and Mass Spectrometry.

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