Distinguishing small molecular mass differences of proteins by mass spectrometry.
Electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry allows for high-resolution, accurate mass analysis of multiply charged ions of proteins. In the work described here, the ability of ESI-FTICR to distinguish small differences in molecular mass is evaluated. Ubiquitin was used as an internal mass calibration standard to measure the molecular mass of cytochrome c, myoglobin, and several carbonic anhydrase isoforms. Mass calibration was based on the tallest isotopic peak of each ubiquitin charge state. Ubiquitin performed well as an internal standard because its charge states covered the appropriate mass range, interference was minimal, and the tallest peak was easily identified. The peak masses of cytochrome c (12.5 kDa) and myoglobin (17 kDa) were measured to an accuracy of about 0.02 Da (<2ppm). However, errors of 1.0 Da were observed for some individual determinations because of the difficulty in identifying the tallest peak. When the technique was applied to bovine carbonic anhydrase II, even combining data from several charge states did not yield an unequivocal assignment of the tallest peak, resulting in a mass assignment of 29,023.7 or 29,024.7. Similarly, measurements of two isoforms with a mass difference of 1 Da, human carbonic anhydrase I, pI 6.0 and 6.6, yielded overlapping values for the mass of the tallest peak. However, these two isoforms were clearly distinguished by (a) identification of the tallest peak using a measurement of average mass as a guide and (b) comparison of the isotopic peak intensity patterns.
Green
M K
MK
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19711, USA.
Vestling
M M
MM
Johnston
M V
MV
Larsen
B S
BS
D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D002138:Calibration; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D017357:Cyclotrons; D003574:Cytochrome c Group / Q000737:chemistry*; D005583:Fourier Analysis; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008970:Molecular Weight; D009211:Myoglobin / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001059:Apoproteins; D003574:Cytochrome c Group; D007527:Isoenzymes; D009211:Myoglobin; D011506:Proteins; C010857:apomyoglobin; D002256:Carbonic Anhydrases