Reversible Click Chemistry Tag for Universal Proteome Sample Preparation for Top-Down and Bottom-Up Analysis

Biedka, Stephanie; Schmidt, Brigitte F.; Frey, Nolan; Boothman, Sarah; Minden, Jonathan S.; Lucas, Amber

doi:10.1021/acs.jproteome.1c00443

Cited by 8 publications

(4 citation statements)

References 46 publications

(62 reference statements)

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“…A protein solution containing sodium dodecyl sulphate (SDS) and dithiothreitol (DTT) was loaded into filtering columns and washed exhaustively with 8 M urea in HEPES buffer. Proteins were then incubated overnight with trypsin sequencing grade (Promega, Madison, USA) after reduction with DTT and alkylation with iodoacetamide (IAA) [ 25 ]. The digested peptides (supernatant) were collected by centrifuging for 20 min at 14,000 rpm at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

Influence of Aldehyde Dehydrogenase Inhibition on Stemness of Endometrial Cancer Stem Cells

Serambeque,

Mestre,

Correia-Barros

et al. 2024

Cancers

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Endometrial cancer is one of the most common gynaecological malignancies. Although often diagnosed at an early stage, there is a subset of patients with recurrent and metastatic disease for whom current treatments are not effective. Cancer stem cells (CSCs) play a pivotal role in triggering tumorigenesis, disease progression, recurrence, and metastasis, as high aldehyde dehydrogenase (ALDH) activity is associated with invasiveness and chemotherapy resistance. Therefore, this study aimed to evaluate the effects of ALDH inhibition in endometrial CSCs. ECC-1 and RL95-2 cells were submitted to a sphere-forming protocol to obtain endometrial CSCs. ALDH inhibition was evaluated through ALDH activity and expression, sphere-forming capacity, self-renewal, projection area, and CD133, CD44, CD24, and P53 expression. A mass spectrometry-based proteomic study was performed to determine the proteomic profile of endometrial cancer cells upon N,N-diethylaminobenzaldehyde (DEAB). DEAB reduced ALDH activity and expression, along with a significant decrease in sphere-forming capacity and projection area, with increased CD133 expression. Additionally, DEAB modulated P53 expression. Endometrial cancer cells display a distinct proteomic profile upon DEAB, sharing 75 up-regulated and 30 down-regulated proteins. In conclusion, DEAB inhibits ALDH activity and expression, influencing endometrial CSC phenotype. Furthermore, ALDH18A1, SdhA, and UBAP2L should be explored as novel molecular targets for endometrial cancer.

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Section: Methodsmentioning

confidence: 99%

Influence of Aldehyde Dehydrogenase Inhibition on Stemness of Endometrial Cancer Stem Cells

Serambeque,

Mestre,

Correia-Barros

et al. 2024

Cancers

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“…Tetrazine ligation has emerged as a powerful bioorthogonal labeling reaction that is distinguished for its speed, selectivity, and tolerance of a wide range of functional groups. − Among their numerous applications in chemical biology, tetrazines have been used as bioorthogonal handles in proteomic workflows. − Recently, the Carrillo group has demonstrated that 3,6-dialkylthiotetrazines can undergo dynamic nucleophilic substitution reactions and that post-functionalization through Diels–Alder reaction with norbornene gives dihydropyridazine products that no longer exchange. , Applications to the syntheses of macrocycles, macromolecular cages, and to polymer chemistry were described, but applications in biology had not been reported . Recently, our group has described a method for preparing thiomethyltetrazines via a one-pot reaction between carboxylic esters and methylthiocarbohydrazide iodide .…”

Section: Introductionmentioning

confidence: 99%

Thiomethyltetrazines Are Reversible Covalent Cysteine Warheads Whose Dynamic Behavior can be “Switched Off” via Bioorthogonal Chemistry Inside Live Cells

Tallon

West

et al. 2023

J. Am. Chem. Soc.

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Electrophilic small molecules that can reversibly modify proteins are of growing interest in drug discovery. However, the ability to study reversible covalent probes in live cells can be limited by their reversible reactivity after cell lysis and in proteomic workflows, leading to scrambling and signal loss. We describe how thiomethyltetrazines function as reversible covalent warheads for cysteine modification, and this dynamic labeling behavior can be "switched off" via bioorthogonal chemistry inside live cells. Simultaneously, the tetrazine serves as a bioorthogonal reporter enabling the introduction of tags for fluorescent imaging or affinity purification. Thiomethyltetrazines can label isolated proteins, proteins in cellular lysates, and proteins in live cells with second-order rate constants spanning 2 orders of magnitude (k 2 , 1−100 M −1 s −1 ). Reversible modification by thiomethyltetrazines can be switched off upon the addition of trans-cyclooctene in live cells, converting the dynamic thiomethyltetrazine tag into a Diels−Alder adduct which is stable to lysis and proteomic workflows. Time-course quenching experiments were used to demonstrate temporal control over electrophilic modification. Moreover, it is shown that "locking in" the tag through Diels−Alder chemistry enables the identification of protein targets that are otherwise lost during sample processing. Three probes were further evaluated to identify unique pathways in a live-cell proteomic study. We anticipate that discovery efforts will be enabled by the trifold function of thiomethyltetrazines as electrophilic warheads, bioorthogonal reporters, and switches for "locking in" stability.

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“…Glycomics investigations commonly rely on sophisticated techniques such as hydrophilic interaction chromatography (HILIC), polymeric anion exchange (WAX), and porous graphitic carbon (PGC) to purify complex glycans [ 4 , 5 ] and mass spectrometry (MS) [ 6 ] to detect desired glycan-containing fractions, and it is further challenging to perform chemical modifications of the selected glycans to facilitate biological studies [ 7 ]. In contrast, the field of proteomics has brought about innovative [ 8 ], selective, mild [ 9 ], and occasionally reversible modifications [ 10 ] of proteins enabling site-specific tagging [ 11 ] and pull-down approaches [ 12 ]. Therefore, the field of proteomics has rapidly progressed with assigning structures and functions to an ever-increasing number of targets [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

Expanding Glycomic Investigations through Thiol-Derivatized Glycans

2023

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N-(2-thioethyl)-2-aminobenzamide (TEAB), a novel glycan auxiliary, was synthesized and its utility was evaluated. The auxiliary was conjugated to glycans by reductive amination with the water-stable reagent 2-picoline borane complex. Glycan products, which ranged from 1 to 7 linked hexoses, were all isolated in yields ranging from 60% to 90% after purification by reverse-phase chromatography. The novel conjugate introduces a convenient, shelf-stable thiol directly onto the desired free glycans with purification advantages and direct modification with efficient reactions through alkenes, halides, epoxides, disulfides, and carboxylates in yields of 49% to 93%. Subsequently, a thiol-selective modification of the BSA protein was used to generate a neoglycoprotein with a bifunctional PEG–maleimide linker. To further illustrate the utility of a thiol motif, 2-thiopyridine activation of a thiol-containing support facilitated the covalent chromatographic purification of labeled glycans in yields up to 63%. Finally, initial proof of concept of implementation in a light printed microarray was explored and validated through FITC-labeled concanavalin A binding. In conclusion, the thiol-functionalized glycans produced greatly expand the diversity of bioconjugation tools that can be developed with glycans and enable a variety of biological investigations.

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Reversible Click Chemistry Tag for Universal Proteome Sample Preparation for Top-Down and Bottom-Up Analysis

Cited by 8 publications

References 46 publications

Influence of Aldehyde Dehydrogenase Inhibition on Stemness of Endometrial Cancer Stem Cells

Influence of Aldehyde Dehydrogenase Inhibition on Stemness of Endometrial Cancer Stem Cells

Thiomethyltetrazines Are Reversible Covalent Cysteine Warheads Whose Dynamic Behavior can be “Switched Off” via Bioorthogonal Chemistry Inside Live Cells

Expanding Glycomic Investigations through Thiol-Derivatized Glycans

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