Dataset Information

Label-free Quantification of Host-Cell Protein Impurity in a Recombinant Hemoglobin Reference Material

ABSTRACT: Quantitative analysis depends on pure-substance primary calibrators with known mass fractions of impurity. The datasets included were generated using mass spectrometry for the evaluation of label-free quantification (LFQ) as a method for determining the mass fraction of host-cell proteins (HCPs) in bioengineered proteins. For this purpose, hemoglobin-A2 (HbA2) was used, as obtained by overexpression in E.coli. Two different materials had been produced: natural, and U-15N-labeled HbA2. To quantify impurity, precursor ion (MS1-) intensities were integrated over all E.coli-proteins identified and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for an E.coli-cell lysate, that had been spiked at known mass-ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and were found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of nine proteins.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human) Escherichia Coli Saccharomyces Cerevisiae (baker's Yeast)

DISEASE(S): Disease Free

SUBMITTER: Cristian Arsene

LAB HEAD: Cristian Arsene

PROVIDER: PXD041736 | Pride | 2023-05-09

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	HbA2_Ecoli_lysate.xlsx	Xlsx
	HbA2_Ecoli_lysate.zip	Other
	HbA2_Ecoli_lysate_identified_proteins.xlsx	Xlsx
	HbA2_Ecoli_lysate_identified_proteins_normalized.xlsx	Xlsx
	HbA2_Ecoli_lysate_normalized.xlsx	Xlsx

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Publications

Label-free quantification of host cell protein impurity in recombinant hemoglobin materials.

Henrion André A Arsene Cristian-Gabriel CG Liebl Maik M O'Connor Gavin G

Analytical and bioanalytical chemistry 20231126 2

Quantitative analysis relies on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host cell proteins (HCPs) in bioengineered proteins which are intended for use as protein calibration standards. In this study a purified hemoglobin-A2 (HbA<sub>2</sub>) protein, obtained through its overexpression in E. coli, was used. Two different materials ...[more]

PMID: 38008782

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