| The combination of electrospray ionization (ESI) and Fourier-transform mass spectrometry (FTMS) is shown to be an accurate and sensitive analytical technique for identification, structural characterization and quantification of biomolecules such as proteins and phospholipids. With its uniquely high resolving power, ESI/FTMS enables analysis of complex mixtures that contain >100 components; and extensive ion fragmentation capabilities.; Top down approach using tandem mass spectrometry provides accurate and efficient identification and structural characterization for proteins <50 kDa. This is demonstrated for characterization of the covalent complex of ThiG (27 kDa) with its substrate analog DDXP, localizing the modification site to Lys96. This was also applied to characterize the disulfide connectivity of structural intermediates of the onconase (12 kDa) oxidative folding.; For proteins larger than 50 kDa, unusually stable noncovalent tertiary structures in the gas phase have made difficult the tandem MS techniques inside the analyzer cell. Initial inhibition of tertiary structure formation with immediate nozzle-skimmer dissociation efficiently improved dissociation of ubiquitin ions, providing backbone cleavages at 74 out of 75 interresidue bonds. Applying such "prefolding dissociation" to far larger proteins, together with addition of conformer disrupting ESI solution additives, the unusual intractability of large protein ions can be reduced with electrospray additives, heated vaporization, and separate noncovalent and covalent bond dissociation to provide extensive information on sequence and posttranslational modifications. Such dissociative top down approach cleaved 287 interresidue bonds in the termini of a 144 kDa protein, specified previously unidentified disulfide bonds between 8 of 27 cysteines in a 200 kDa protein, and corrected sequence predictions in two proteins, one with 229 kDa.; ESI/MS was also applied to quantitative analysis of ∼100 phospholipid components of detergent resistant membranes from RBL-2H3 cells, characterizing the signaling requirements and kinetics for the change in the lipid composition, which is manifested as an increase in polyunsaturated phospholipids. We find that this change can be effectively prevented by an F-actin stabilizer, jasplakinolide; whereas F-actin disrupters, such as cytochalasin D, rapidly elicit the similar change in the lipid composition of in the absence of antigen stimulation, indicating that perturbations in the actin cytoskeleton affect the organization of plasma membrane lipid rafts. |
