Understanding the influence of post-excite radius and axial confinement on quantitative proteomic measurements using Fourier transform ion cyclotron resonance mass spectrometry.
Early studies of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) explored many of the fundamental issues surrounding the potential of the technique to provide quantitative data. Improvements in instrument technology and the analysis of larger molecules in increasingly complex mixtures warrant not only a revisit to some of these earlier studies, but a more comprehensive examination of the influence of various instrument parameters on quantitative (absolute and relative) measurements in proteomics. We present a detailed examination of the role that acquisition time, excite voltage (i.e. excite radius), trapping voltage, and the type of excitation waveform have on the ability of FT-ICR to accurately quantify biological molecules. The use of a stable-isotope-labeled and unlabeled phenyl isocyanate derivatized peptide allows us to ascribe the effects of FT-ICR-MS on quantification, thus eliminating the contribution of ionization differences to ion abundance. To adequately assess the multiple parameters in the large dataset, we develop a multiplicative quality factor that encompasses the total ion abundance, as well as the accuracy and the precision of abundance ratios. This assessment allows facile determination of optimal instrument parameters for quantitative measurements.
Frahm
Jennifer L
JL
Department of Chemistry, North Carolina State University, Dabney Hall, Campus Box 8204, Raleigh, NC 27695-8204, USA.
Velez
Coral M Capo
CM
Muddiman
David C
DC
D016477:Artifacts; D017357:Cyclotrons; D007477:Ions; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D007477:Ions; D020543:Proteome