Trastuzumab is known to be heterogeneous in terms of charge. Stressing trastuzumab under physiological conditions (pH 7.4 and 37 degrees C) increases charge heterogeneity further. Separation of... Show moreTrastuzumab is known to be heterogeneous in terms of charge. Stressing trastuzumab under physiological conditions (pH 7.4 and 37 degrees C) increases charge heterogeneity further. Separation of charge variants of stressed trastuzumab at the intact protein level is challenging due to increasing complexity making it difficult to obtain pure charge variants for further characterization. Here we report an approach for revealing charge heterogeneity of stressed trastuzumab at the subunit level by pH gradient cation-exchange chromatography. Trastuzumab subunits were generated after limited proteolytic cleavage with papain, IdeS, and GingisKHAN (R). The basic pI of Fab and F(ab)(2) fragments allowed to use the same pH gradient for intact protein and subunit level analysis. Baseline separation of Fab subunits was obtained after GingisKHAN (R) and papain digestion and the corresponding modifications were determined by LC-MS/MS peptide mapping and middle-down MALDI-ISD FT-ICR MS. The described approach allows a comprehensive charge variant analysis of therapeutic antibodies that have two or more modification sites in the Fab region. Show less
Szegedi-Elek, E.; Ábrahám, P.; Wyrzykowski, Ł.; Kun, M.; Kóspál, Á.; Chen, L.; ... ; Ziółkowska, O. 2020
Formaldehyde fixation is widely used for long-term storage of tissue. However, due to formaldehyde-induced cross-links, fixated tissue proteins are difficult to extract hampering mass spectrometry ... Show moreFormaldehyde fixation is widely used for long-term storage of tissue. However, due to formaldehyde-induced cross-links, fixated tissue proteins are difficult to extract hampering mass spectrometry (MS)-proteomic analyses. In the present work we evaluate cleavable protein crosslinkers as fixatives that allow tissue preservation as well as efficient protein extraction for MS-proteomics under mild conditions. We investigated disuccinimidyl tartrate (DST) and dithiobis[succinimidylpropionate] (DSP) as cleavable fixating reagents. These compounds crosslink proteins by reacting with amino groups leading to amide bond formation and can be cleaved with sodium metaperiodate (cis-diols, DST) or reducing agents (disulfide bonds, DSP). Our results show that cleavable protein crosslinking with DST and DSP allows tissue fixation with morphology preservation comparable to formaldehyde.Cleavage of DSP improves protein recovery from fixated tissue by a factor of 18 and increases the number of identified proteins by approximately 20% under mild extraction conditions compared to formaldehyde-fixated paraffin-embedded tissue. A major advantage of DSP is the introduction of well-defined protein modifications that can be taken into account during database searching. In contrast to DSP fixation, DST fixation followed by cleavage with sodium metaperiodate, while effective, resulted in side reactions that prevented effective protein extraction and interfered with protein identification. Show less
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