Observation of heterogeneous gene products by FT-ICR MS.
The ability of FT-ICR MS to resolve isotopic variants of intact proteins for each of the charge states formed by electrospray ionization offers a sensitive, rapid method for detecting "low mass" heterogeneity, where this is defined as the presence of structural variants differing in mass by 2 Da or less. Such heterogeneity may reflect biological or chemical modifications of structure or may result from the coexpression of related proteins from a multi-gene family. In the analytical approach described here, comparisons are made between observed isotopic distributions and those expected for predicted protein sequences. Close agreement is demonstrated for a homogeneous model protein, and the utility of the method has been evaluated in the study of mouse major urinary proteins (MUPs), a group of closely related sequences. Divergence of the experimental isotopic distribution from distributions predicted for known MUP sequences can be explained, in quantitative terms, by the coexpression of closely related sequences. This approach provides a facile method for the assessment of protein homogeneity and for the detection of structural variants, without recourse to proteolytic digestion and analysis of the resulting products.
Robertson
Duncan H L
DH
Protein Function Group, Faculty of Veterinary Science, University of Liverpool, UK. dhlr@liverpool.ac.uk
Wong
Stephen C C
SC
Beynon
Robert J
RJ
Hurst
Jane L
JL
Gaskell
Simon J
SJ
D000818:Animals; D006736:Horses; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009211:Myoglobin / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D053719:Tandem Mass Spectrometry
D009211:Myoglobin; D011506:Proteins; C035737:major urinary proteins