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

Electrophoresis. 2000 Jan;21(2):430-9. doi: 10.1002/(SICI)1522-2683(20000101)21:2<430::AID-ELPS430>3.0.CO;2-0.


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 degrees 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.

MeSH terms

  • Animals
  • Cattle
  • Electrophoresis, Gel, Two-Dimensional / methods*
  • Humans
  • Mass Spectrometry* / instrumentation
  • Mass Spectrometry* / methods
  • Protein Conformation
  • Proteins / analysis*
  • Proteins / chemistry


  • Proteins