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 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.
Kristensen
D B
DB
Hiroshima Tissue Regeneration Project, Hiroshima Prefectural Institute of Industrial Science and Technology, Higashihiroshima, Japan.
Imamura
K
K
Miyamoto
Y
Y
Yoshizato
K
K
D000818:Animals; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis* / Q000737:chemistry
D011506:Proteins