Electron capture dissociation for structural characterization of multiply charged protein cations.

For proteins of < 20 kDa, this new radical site dissociation method cleaves different and many more backbone bonds than the conventional MS/MS methods (e.g., collisionally activated dissociation, CAD) that add energy directly to the even-electron ions. A minimum kinetic energy difference between the electron and ion maximizes capture; a 1 eV difference reduces capture by 10(3). Thus, in an FTMS ion cell with added electron trapping electrodes, capture appears to be achieved best at the boundary between the potential wells that trap the electrons and ions, now providing 80 +/- 15% precursor ion conversion efficiency. Capture cross section is dependent on the ionic charge squared (z2), minimizing the secondary dissociation of lower charge fragment ions. Electron capture is postulated to occur initially at a protonated site to release an energetic (approximately 6 eV) H. atom that is captured at a high-affinity site such as -S-S- or backbone amide to cause nonergodic (before energy randomization) dissociation. Cleavages between every pair of amino acids in mellitin (2.8 kDa) and ubiquitin (8.6 kDa) are represented in their ECD and CAD spectra, providing complete data for their de novo sequencing. Because posttranslational modifications such as carboxylation, glycosylation, and sulfation are less easily lost in ECD than in CAD, ECD assignments of their sequence positions are far more specific.

Heterogeneous glycosylation of immunoglobulin E constructs characterized by top-down high-resolution 2-D mass spectrometry.

Posttranslational glycosylation is critical for biological function of many proteins, but its structural characterization is complicated by natural heterogeneity, multiple glycosylation sites, and different forms. Here, a top-down mass spectrometry (MS) characterization is applied to three constructs of the Fc segment of IgE: Fcepsilon(3-4) (52 kDa) and Fcepsilon(2-3-4)(2) (76 kDa) disulfide-bonded homodimers. Fourier transform MS of a reduced sample of Fcepsilon(2-3-4) gave molecular masses of 37 527, 37 689, 37 851, and 38 014 Da, directly characterizing multiple glycoforms (hexose = 162 Da) without chromatographic separation. Limited proteolysis of the nonreduced Fcepsilon(2-3-4)(2) protein yielded a peptide mixture with molecular weight values that agreed with those expected from the DNA sequence. The single glycosylation site in these constructs was identified, and quantities were determined of five glycoforms that agreed within +/-2% of the molecular ion values. The 2-D mass spectrum of two glycosylated peptides showed these to have high-mannose structures, -GlcNAc-(hex)(n)(), demonstrating that Fcepsilon(2-3-4) has a single such structure of n = 5-9. For a mutated sample of Fcepsilon(3-4), in addition to five glycoforms, MS showed a molecular discrepancy that could be assigned with proteolysis and 2-D mass spectra to the oxidation of two methionines and an additional residue difference.

Quantitative analysis of phospholipids in functionally important membrane domains from RBL-2H3 mast cells using tandem high-resolution mass spectrometry.

We recently showed that ligand-mediated cross-linking of FcepsilonRI, the high-affinity receptor for immunoglobulin E, on RBL-2H3 mast cells results in its co-isolation with detergent-resistant membranes (DRM) and its consequent tyrosine phosphorylation by the co-localized tyrosine kinase Lyn that is a critical early event in signaling by this receptor [Field et al. (1997) J. Biol. Chem. 272, 4276-4280]. As part of efforts to determine the structural bases for these interactions, we examined the phospholipid composition of DRM vesicles isolated from RBL-2H3 cells under conditions that preserve FcepsilonRI association. We used positive and negative mode electrospray Fourier transform ion cyclotron resonance mass spectrometry to compare quantitatively the phospholipid composition of isolated DRM to that of total cell lipids and to a plasma membrane preparation. From these analyses, over 90 different phospholipid species were spectrally resolved and unambiguously identified; more than two-thirds of these were determined with a precision of +/-0.5% (absolute) or less. Quantitative characterization of lipid profiles shows that isolated DRM are substantially enriched in sphingomyelin and in glycerophospholipids with a higher degree of saturation as compared to total cellular lipids. Plasma membrane vesicles isolated from RBL-2H3 cells by chemically induced blebbing exhibit a degree of phospholipid saturation that is intermediate between DRM and total cellular lipids, and significant differences in the headgroup distribution between DRM and plasma membranes vesicles are observed. DRM from cells with cross-linked FcepsilonRI exhibit a larger ratio of polyunsaturated to saturated and monounsaturated phospholipids than those from unstimulated cells. Our results support and strengthen results from previous studies suggesting that DRM have a lipid composition that promotes liquid-ordered structure. Furthermore, they demonstrate the potential of mass spectrometry for examining the role of membrane structure in receptor signaling and other cellular processes.

Electrospray mass spectra from protein electroeluted from sodium dodecylsulfate polyacrylamide gel electrophoresis gels.

Electrospray ionization/tandem mass spectrometry of proteins separated on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels is severely limited by the requirement that the protein be completely separated from the SDS. As shown here, the gaseous noncovalent SDS adducts of protonated proteins thus formed can be dissociated by infrared irradiation. ESI spectra from myoglobin in SDS-containing solutions show molecular ions adducted with up to 15 dodecyl sulfates, but ir irradiation of these ions causes complete dissociation to expel the SDS.

Two-dimensional mass spectrometry of biomolecules at the subfemtomole level.

Multiple dimensions of unique molecular structure information can now be obtained from proteins and DNA using mass spectrometry. Less than 10(-16) mol of the active major histocompatibility complex signaling peptide in a mixture of thousands can be identified. For large proteins (> 40 kDa), the high resolving power (> 10(5) and 10(-17) mol sensitivity of Fourier-transform mass spectrometry provide exact molecular weight values (+/- 1 or 2 Da) for mixture components, indicating error or modifications compared with the predicted DNA sequence. Selecting a specific molecular species, its two-dimensional spectrum indicates the part of the molecule that is modified; a three-dimensional spectrum of that fragment further isolates the modification site.