A rapid discriminative hydrogen–deuterium exchange and LC-HRMS/MS strategy for primary and higher order structural mapping of therapeutic proteins: a case study using filgrastim

Thakkar, Harsh; Eerla, Rameswari; Sharma, Lokesh; Shah, Ravi P.

doi:10.1039/d2ay01788a

Cited by 1 publication

(2 citation statements)

References 18 publications

(17 reference statements)

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“…HDX-MS was performed similarly to our previous work . Each time filgrastim biosimilar was checked as system suitability for HDX-MS methodology.…”

Section: Methodsmentioning

confidence: 99%

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Cell-Engineered Recombinant α-Synuclein: A Gage R&R Validated Protocol

Thakkar,

Chatterjee,

Saxena

et al. 2023

J. Proteome Res.

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α-Synuclein (α-Syn) misfolding and its presence in Lewy bodies are observed in almost all Parkinson's disease (PD) patients. Basic biomedical research would benefit from a quick, low-cost approach to purifying α-Syn and developing in vitro and in vivo models for PD. Several research groups utilize PFF-based models, yet the production of α-Syn PFFs is inconsistent, resulting in nonconclusive findings. Some research laboratories prepare recombinant α-Syn (r α-Syn) by molecular cloning to overexpress α-Syn with various purifying techniques. Laboratory-to-laboratory protocols cause considerable variability and sometimes contradictory findings. PD researchers spend more on protein than solving α-Syn's riddles. This article uncovered a novel method for expressing and purifying r α-Syn validated through gage reproducibility and repeatability (Gage R&R). For the production of r α-Syn, we have employed the ability of a high-cell-density-based expression system to overexpress protein in BL21(DE3). A simple, high-throughput, nonchromatographical purification protocol has been devised to facilitate research with higher reproducibility, which was validated through Gage R&R. A crossover experimental design was utilized, and the purified protein was characterized using orthogonal high-end analytical methods, which displayed higher similarity between the isolated r α-Syn. Batch-to-batch variability was the least for produced protein and hence can be utilized for exploring the iceberg of PD.

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“…HDX-MS was performed similarly to our previous work . Each time filgrastim biosimilar was checked as system suitability for HDX-MS methodology.…”

Section: Methodsmentioning

confidence: 99%

“…HDX-MS was performed similarly to our previous work. 16 Each time filgrastim biosimilar was checked as system suitability for HDX-MS methodology. Five micrograms of isolated protein was incubated in deuterium oxide for 3 h, and the samples were injected into the system.…”

Section: Analytical Characterization Of Gage Randr Batchesmentioning

confidence: 99%