Sensitivity booster for mass detection enables unambiguous analysis of peptides, proteins, antibodies, and protein bioconjugates

Singudas, Rohith; Reddy, Neelesh C; Rai, Vishal

doi:10.1039/c9cc03424b

Cited by 13 publications

(17 citation statements)

References 16 publications

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“…Alongside this, the drug 9 e (DM1) was conveniently installed on mAb 13 to render the ADC (15, Figure 5). Further, the results of the MTT assay confirm the dose-dependent response of ADC (15) in HER-2 overexpressing SKBR-3 breast cancer cells. The LDM K-K ADC 15 showed 45 % growth inhibition at 2 nm concentration in comparison to 38 % with Kadcyla (16) in SKBR-3 cells (Figure 5 b).…”

Section: Angewandte Chemiesupporting

confidence: 61%

“…Next, we selected ubiquitin (1 d) with one N a -NH 2 and seven N e -NH 2 groups. The LDM K-K reagents 4 a and 4 c-4 f led to 15,10,8,13, and 5 % conversion, respectively (Figures S47-S51). To our delight, the LDM K-K reagent 4 b proved successful in rendering single-site (K6) labeled ubiquitin 6 d (35 % conv., Figures 3 and S27).…”

Section: Angewandte Chemiementioning

confidence: 98%

“…After successful demonstration of LDM K-K with proteins bearing moderate number of primary amines, we selected a-lactalbumin, cytochrome C, and myoglobin with higher number of primary amines (13)(14)(15)(16)(17)(18)(19)(20). The vortexing of a-lactalbumin 1 e (one N a -NH 2 and twelve N e -NH 2 ) with 4 a renders mono-labeled product 6 e (37 % conv., Figures 3 and S28).…”

Section: Angewandte Chemiementioning

confidence: 99%

“…The SDS-PAGE exhibits the fluorescent band for both heavy and light chains (Figure S57 b). Our in-house sensitivity booster technology [15] enabled an unambiguous analysis of the sites-of-conjugation (K169-light chain and K395-heavy chain, Figure S59). Alongside this, the drug 9 e (DM1) was conveniently installed on mAb 13 to render the ADC (15, Figure 5).…”

Section: Angewandte Chemiementioning

confidence: 99%

“…However, the off-target reactions in protein modification resulting in a heterogeneous mixture of products remains a significant concern. [15] Primarily, it is due to the non-trivial task of identifying a single residue amongst a range of nucleophilic residues. Further, the diverse microenvironment and solvent accessibility of proteinogenic residues enhance the complexity.…”

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confidence: 99%

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Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

et al. 2020

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The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single‐site labeling of a high‐frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent‐accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys‐selective electrophile connected by a spacer. Consequently, it enables the irreversible single‐site labeling of a Lys residue independent of its place in the reactivity order. The user‐friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site‐selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe‐tagged proteins. Besides, the methodology provides access to antibody‐drug conjugate (ADC), which exhibits highly selective anti‐proliferative activity towards HER‐2 expressing SKBR‐3 breast cancer cells.

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Section: Angewandte Chemiesupporting

confidence: 61%

Section: Angewandte Chemiementioning

confidence: 98%

Section: Angewandte Chemiementioning

confidence: 99%

Section: Angewandte Chemiementioning

confidence: 99%

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confidence: 99%

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Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

et al. 2020

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One‐Step Azolation Strategy for Site‐ and Chemo‐Selective Labeling of Proteins with Mass‐Sensitive Probes

Tang

Raj

2020

Angewandte Chemie

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The chemical modification of proteins in as iteselective manner leads to many advances in various scientific fields.T he major challenges with conventional N-terminal bioconjugation techniques are the lacko fu niversal sequence compatibility and poor mass-detection sensitivity of the resulting bioconjugates.T his approach efficiently analyzes proteolytic fragments and native proteins in ac omplex mixture. Multiple chemical steps are usually required for the siteselective synthesis of bioconjugates with enhanced massdetection sensitivity.W epresent asingle-step,versatile strategy for the selective modification of protein N-termini with mass boosters.T he chemical tag enhances the peptide detection by multiple orders thus leading to the unambiguous analysis of the resulting bioconjugates.W ed emonstrate that tagging proteolytic fragments with mass sensitivity probes in ac omplex mixture improves the detection of resulting bioconjugates.

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A Tag‐Free Platform for Synthesis and Screening of Cyclic Peptide Libraries

Bruce,

Adebomi,

Czabala

et al. 2024

Angewandte Chemie

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In the realm of high‐throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post‐screening. Our innovation introduces a tag‐free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick Chemistries (CCDC) discovered within our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity‐enhancing derivatization method, utilized in tandem with nano LC‐MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we've successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV CAPSID protein responsible for viral infections as validated by microscale thermalshift assays (TSA), biolayer Interferometry (BLI) and functional assays.

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Sensitivity booster for mass detection enables unambiguous analysis of peptides, proteins, antibodies, and protein bioconjugates

Abstract: A chemical tag enhances peptide detection by multiple orders in mass spectrometry.

Cited by 13 publications

References 16 publications

Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

One‐Step Azolation Strategy for Site‐ and Chemo‐Selective Labeling of Proteins with Mass‐Sensitive Probes

A Tag‐Free Platform for Synthesis and Screening of Cyclic Peptide Libraries

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