Rapid Measurement of Protein Modifications by Combining Exact Mass Measurement MS and Advanced Spectral Deconvolution

Library Number:
Part Number:
Beth L. Gillece-Castro;Ziling Lu;Thomas E. Wheat;and Jeffrey R. Mazzeo [Waters]
Second Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology and Pharmaceutical Industries, Practical MS
Content Type:
Content Subtype:
Practical MS
Detecting and measuring modifications in protein structure that affect biological activity is required in protein chemistry research. For protein biopharmaceuticals, such changes must be monitored throughout process development, stability testing, and quality assurance because structural changes can affect efficacy and safety. While peptide mapping can be used, it is time-consuming and labor-intensive. It is attractive to consider using molecular weight measurement of the intact protein to determine the degree of modification. Such an approach would require measurement of exact mass in combination with spectral interpretation that preserves quantitative information. For this approach to be used, high quality, exact mass spectra are required. Of equal importance, the spectra must be analyzed using an algorithm that does not require prior knowledge of the sample composition and that preserves relative quantitative information about the protein components of the sample. An oa-Tof instrument, LCT Premier, in conjunction with the MaxEnt1algorithm meets these basic criteria. In these experiments, we have explored the power and limitations of this combination. The simple procedure for determining MaxEnt1 parameter settings will be described in detail, together with the effect of deviations. We will show the steps in evaluating the quality of the deconvolution result without prior knowledge of the true result. Using the systematic setup, the magnitude of MW changes that can be detected and the dynamic range properties of the analysis will be presented. In addition, we will show the optimization of the analysis for the changes likely to be encountered in stability testing, such as, oxidation. The approach will also be applied to measuring variability in glycosylation state of recombinant proteins

Title Format File Size
wa43189 PDF 464.59kB