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1.
Anal Chem ; 95(4): 2532-2539, 2023 01 31.
Article in English | MEDLINE | ID: mdl-36652389

ABSTRACT

The development of MS-cleavable cross-linking mass spectrometry (XL-MS) has enabled the effective capture and identification of endogenous protein-protein interactions (PPIs) and their residue contacts at the global scale without cell engineering. So far, only lysine-reactive cross-linkers have been successfully applied for proteome-wide PPI profiling. However, lysine cross-linkers alone cannot uncover the complete PPI map in cells. Previously, we have developed a maleimide-based cysteine-reactive MS-cleavable cross-linker (bismaleimide sulfoxide (BMSO)) that is effective for mapping PPIs of protein complexes to yield interaction contacts complementary to lysine-reactive reagents. While successful, the hydrolysis and limited selectivity of maleimides at physiological pH make their applications in proteome-wide XL-MS challenging. To enable global PPI mapping, we have explored an alternative cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide (DBrASO). Our results have demonstrated that DBrASO cross-linked peptides display the same fragmentation characteristics as other sulfoxide-containing MS-cleavable cross-linkers, permitting their unambiguous identification by MSn. In combination with a newly developed two-dimensional peptide fractionation method, we have successfully performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated its capability to complement lysine-reactive reagents and expand PPI coverage at the systems-level.


Subject(s)
Cysteine , Proteome , Humans , Proteome/chemistry , Lysine , HEK293 Cells , Peptides/chemistry , Mass Spectrometry/methods , Sulfoxides/chemistry , Cross-Linking Reagents/chemistry
2.
Org Lett ; 24(5): 1190-1194, 2022 02 11.
Article in English | MEDLINE | ID: mdl-35094508

ABSTRACT

We report the first total syntheses of strasseriolide A and B. Strasseriolide B shows potent activity against the wild-type malaria parasite Plasmodium falciparum and good activity against a chloroquine-resistant strain. A convergent strategy was envisioned with an aldehyde-acid fragment and a vinyl iodide-alcohol fragment. Both fragments were prepared using chiral pool starting materials. They were combined with a Yamaguchi esterification and cyclized with a Nozaki-Hiyama-Kishi reaction. Strasseriolide B was assembled in a 16-step LLS.


Subject(s)
Antimalarials , Biological Products , Macrolides , Plasmodium falciparum , Antimalarials/chemical synthesis , Antimalarials/chemistry , Antimalarials/pharmacology , Biological Products/chemical synthesis , Biological Products/chemistry , Biological Products/pharmacology , Macrolides/chemical synthesis , Macrolides/chemistry , Macrolides/pharmacology , Molecular Conformation , Parasitic Sensitivity Tests , Plasmodium falciparum/drug effects
3.
Mol Cell Proteomics ; 20: 100084, 2021.
Article in English | MEDLINE | ID: mdl-33915260

ABSTRACT

Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions nontargetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions because of nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MSn. The MSn-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.


Subject(s)
Cross-Linking Reagents/chemistry , Diazomethane/analogs & derivatives , Diazomethane/chemistry , Chromatography, Liquid , Fungal Proteins/chemistry , Mass Spectrometry/methods , Photochemical Processes , Proteasome Endopeptidase Complex/chemistry , Saccharomyces cerevisiae , Serum Albumin, Bovine
4.
Photochem Photobiol ; 93(2): 473-478, 2017 03.
Article in English | MEDLINE | ID: mdl-27696431

ABSTRACT

In the southern Appalachian area of the United States, the Phausis reticulata firefly, commonly known as the "Blue Ghost," performs a unique display of bioluminescence. Adult male organisms are observed darting rapidly along paths and riverbeds in dark forests producing long-lasting and mesmerizing bluish-white luminous streaks. Starting with eighteen adult male firefly lanterns, we used a reverse transcriptase and rapid amplification of cDNA ends (RACE) approach to clone the 1635 base pair open reading frame of the P. reticulata luc gene corresponding to a 545 residue protein. Expression of the recombinant luciferase protein in Escherichia coli and characterization studies revealed the true color of the light emission to be green (λmax = 552 nm), strongly suggesting that the field observations result from a Purkinje shift. While the P. reticulata luciferase amino acid sequence is 74.3% identical to the North American Photinus pyralis luciferase, we were surprised to find that it was 88.4% and 87.7% identical to luciferases from C. ruficollis and D. axillaris both native to mainland Japan. Phylogenetic analysis confirmed the close relationship of the three enzymes that is surprising given the great distance between their natural habitats and the inability of the Japanese fireflies to produce bright bioluminescence.


Subject(s)
Luciferases, Firefly/genetics , Luminescence , Amino Acid Sequence , Animals , Appalachian Region , Base Pairing , Cloning, Molecular , Escherichia coli/genetics , Fireflies/classification , Luciferases, Firefly/chemistry , Luciferases, Firefly/metabolism , Male , Open Reading Frames , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction
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