Published:
Springer Science and Business Media LLC, 2024
Published in:
Analytical and Bioanalytical Chemistry, 416 (2024) 2, Seite 387-396
Language:
English
DOI:
10.1007/s00216-023-05024-8
ISSN:
1618-2642;
1618-2650
Origination:
Footnote:
Description:
AbstractQuantitative 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 (HbA2) protein, obtained through its overexpression in E. coli, was used. Two different materials were produced: natural and U15N-labeled HbA2. For the quantification of impurities, 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, which 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 found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of just nine proteins, and a minimum of five proteins was estimated to be sufficient to keep the sampling error below 15%. For the studied materials, HbA2 mass fractions (or purities) of 923 and 928 mg(HbA2)/g(total protein) were found with expanded uncertainties (U) of 2.8 and 1.3%, resp. Value assignment by LFQ thus contributes up to about 3% of the overall uncertainty of HbA2 quantification when these materials are used as calibrators. Further purification of the natural HbA2 yielded a mass fraction of 999.1 mg/g, with a negligible uncertainty (U = 0.02%), though at a significant loss of material. If an overall uncertainty of 5% is acceptable for protein quantification, working with the original materials would therefore definitely be viable, circumventing the need of further purification.