Mass spectrometric approaches for the characterization of proteins on a hybrid quadrupole time‐of‐flight (Q‐TOF) mass spectrometer

This study demonstrates structural and conformational characterization of proteins by nanoflow electrospray ionization (nanoESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) utilizing a quadrupole time‐of‐flight (Q‐TOF) mass spectrometer (Micromass, Manchester, England). Model peptides were successfully sequenced at the 35 attomole (amol) level, and peptides derived from a tryptic in‐gel digest of 25 femtomole (fmol) bovine serum albumin (BSA) were successfully sequenced. The results demonstrated that the MS/MS sensitivity of the Q‐TOF clearly surpassed the detection limit of the silver stain. A silver destaining step greatly improved the mass analysis of peptides derived from in‐gel digests. Interestingly, sequence analysis revealed BSA residue 424 (tyrosine) as a potential chlorination site. In addition, a modified procedure was successfully used to extract and measure the masses of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE)‐resolved proteins in the 10—68.5 kDa range. The Q‐TOF was also used to monitor conformational changes of proteins. These experiments demonstrated an acid‐induced denaturation of BSA in the pH 3—4 range, and heat‐induced unfolding of cytochrome c between 50 and 60°C. Finally, Zn2+ binding was demonstrated for the carbonic anhydrase apoprotein. In summary, the wide range of applications and the high quality of the experimental data made the Q‐TOF mass spectrometer a powerful analytical tool for protein characterization.

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