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Single proteins separated by 2-DE often show multiple spots spreading along the first dimension. In many cases, such charge trains are explained by isoform differences or by putative post-translational modifications including phosphorylation, glycosylation and others. We now report that individual spots of such charge trains on 2-D gels in fact often represent the same protein, but, apparently due to conformational changes, segregate to different isoelectric points. If MS analysis reveals protein identity, we therefore suggest integrating all individual spots within a charge train for quantification. Especially in quality control of pharmaceutical proteins, the integration of the spot groups of all active contents is preferable in order to obtain reproducible and reasonable quantitative results. However, most commercial software packages for gel analysis integrate the signals spot-wise. We provide an improved quantification tool for proteins with charge train groups. This calculation can be implemented using the MATLAB software and the self-developed "Correct Integration Software System" or the commercial software package Delta2D.

2-DE and MALDI-TOF MS are useful techniques for the quality evaluation of medicinal products derived from recombinant DNA technology. The principal objective of this study has been to evaluate the suitability of 2-DE in combination with MALDI-TOF MS for the quality study of the therapeutic recombinant protein, abatacept. 1-DE SDS-PAGE, under reducing and nonreducing conditions, and 2-DE analysis were used for the assessment of M(r) , pI, and enzymatic deglycosylation efficiency of abatacept. 2-DE allowed the assessment of product identity, purity, charge heterogeneity, isoform pattern, and post-translational modifications. Furthermore, optimization of the deglycosylation procedure, charge heterogeneity, and sample preparation for the subsequent MALDI-TOF MS analysis has been addressed. PMF analysis allowed rapid identity confirmation of abatacept.