MALDI In-Source Decay of High Mass Protein Isoforms: Application to α- and β-Tubulin Variants

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• 作者：David Calligaris ; Claude Villard ; Lionel Terras ; Diane Braguer ; Pascal Verdier-Pinard ; Daniel Lafitte
• 刊名：Analytical Chemistry
• 出版年：2010
• 出版时间：July 15, 2010
• 年：2010
• 卷：82
• 期：14
• 页码：6176-6184
• 全文大小：524K
• 年卷期：v.82,no.14(July 15, 2010)
• ISSN：1520-6882

文摘

Tubulin is one of the major targets in cancer chemotherapy and the target of more than twenty percent of the cancer chemotherapic agents. The modulation of isoform content has been hypothesized as being a cause of resistance to treatment. Isoform differences lie mostly in the C-terminus part of the protein. Extensive characterization of this polypeptide region is therefore of critical importance. MALDI-TOF fragmentation of tubulin C-terminal domains was tested using synthetic peptides. Then, isotypes from HeLa cells were successfully characterized for the first time by in-source decay (ISD) fragmentation of their C-terminus coupled to a pseudo MS³ technique named T³-sequencing. The fragmentation occurred in-source, preferentially generating y_n-series ions. This approach required guanidination for the characterization of the β_III-tubulin C-terminus peptide. This study is, to our knowledge, the first example of reflectron in-source decay (reISD) of the C-terminus of a 50 kDa protein. This potentially occurs via a CID-like mechanism occurring in the MALDI plume. There are now new avenues for top-down characterization of important clinical biomarkers such as β_III-tubulin isotypes, a potential marker of drug resistance and tumor progression. This paper raises the challenge of protein isotypes characterization for early cancer detection and treatment monitoring.