Blas A. Cerda

Electron capture dissociation of gaseous multiply charged ions by fourier-transform ion cyclotron resonance

Fourier-transform ion cyclotron resonance instrumentation is uniquely applicable to an unusual new ion chemistry, electron capture dissociation (ECD). This causes nonergodic dissociation of far larger molecules (42 kDa) than previously observed (<1 kDa), with the resulting unimolecular ion chemistry also unique because it involves radical site reactions for similarly larger ions. ECD is highly complementary to the well known energetic methods for multiply charged ion dissociation, providing much more extensive protein sequence information, including the direct identification of N- versus C-terminal fragment ions. Because ECD only excites the molecule near the cleavage site, accompanying rearrangements are minimized. Counterintuitively, cleavage of backbone covalent bonds of protein ions is favored over that of noncovalent bonds; larger (>10 kDa) ions give far more extensive ECD if they are first thermally activated. This high specificity for covalent bond cleavage also makes ECD promising for studying the secondary and tertiary structure of gaseous protein ions caused by noncovalent bonding.

Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3 mastocytoma cells during degranulation.

Biological membranes contain an extraordinary diversity of lipids. Phospholipids function as major structural elements of cellular membranes, and analysis of changes in the highly heterogeneous mixtures of lipids found in eukaryotic cells is central to understanding the complex functions in which lipids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that granule fusion is initiated by addition of exogenous, nonmammalian phospholipases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to measure changes in the glycerophospholipid composition of total lipid extracts of intact and permeabilized RBL-2H3 (mucosal mast cell line) cells. The low energy of the electrospray ionization results in efficient production of molecular ions of phospholipids uncomplicated by further fragmentation, and changes were observed that eluded conventional detection methods. From these analyses we have spectrally resolved more than 130 glycerophospholipids and determined changes initiated by introduction of exogenous phospholipase C, phospholipase D, or phospholipase A2. These exogenous phospholipases have a preference for phosphatidylcholine with long polyunsaturated alkyl chains as substrates and, when added to permeabilized mast cells, produce multiple species of mono- and polyunsaturated diacylglycerols, phosphatidic acids, and lysophosphatidylcholines, respectively. The patterns of changes of these lipids provide an extraordinarily rich source of data for evaluating the effects of specific lipid species generated during cellular processes, such as exocytosis.

Hydrogen atom loss in electron-capture dissociation: a Fourier transform-ion cyclotron resonance study with single isotopomeric ubiquitin ions

In electron-capture dissociation (ECD), a multiply-protonated protein ion, trapped in a Fourier transform-ion cyclotron resonance (FT-ICR) cell, captures a low-energy electron at a protonated site. In a major reaction pathway, the resulting hydrogen atom attacks a backbone carbonyl oxygen to form a hypervalent species that immediately dissociates into a complementary c, z• ion pair. For larger proteins, the reduced odd-electron ion (M + nH)(n −1)+• is a major product, as shown here using isotopically isolated precursors. In addition, a hydrogen atom can be lost without further reaction, yielding the [M + (n −1)H](n −1)+ even-electron ions. The large effect of charge state on the yield of these ions suggests that the 9+ to 11+ charge states have novel charge-solvated secondary structures.

Electron Capture Dissociation of Multiply-Charged Oxygenated Cations. A Nonergodic Process

Several mechanistic aspects have been proposed as important in causing the unusual ion chemistry induced in multiply-charged protein cations by electron capture. The 5–7 eV energy released by neutralization appears to induce cleavage before energy randomization (nonergodic), and the electron forms radical species whose activation energies for dissociation should be much lower. In contrast, electron capture by [HO(C2H4O)24H + 2H]2+ ions from polyethylene glycol yields no radical ions, losing H• consistent with the lower H• affinity of the hydroxyl and ether groups vs the amide and S–S functionalities of proteins. However, the dominant product ions, [HO(C2H4O)24–nH + H]+ (n = 2 to 8), do appear to be formed by nonergodic dissociation of the hypervalent (M + 2H)1+• intermediate. The expected complementary alkoxy radical ion product is not found, possibly due to an energetic Franck–Condon relaxation. Precursors ionized with (NH4)22+ and Na22+ yield ECD products that are analogous but of different size (n values). Those for Na22+ can be rationalized with structures proposed by Bowers and coworkers. ECD spectra of polyethers should be useful for sequencing.

Charge/radical site initiation versus coulombic repulsion for cleavage of multiply charged ions. Charge solvation in poly(alkene glycol) ions

Electrospray ionization of poly(ethylene glycol) (PEG) followed by separation with Fourier-transform mass spectrometry traps (PEG100 + nH)n+ ions. Both collisionally activated dissociation (CAD) and electron capture dissociation (ECD) of these ions (n = 5, 6, 7) produce PEGx fragment ions in which the x values correspond closely to those for an equal distribution of charges in the linear polymer ion, e.g., for n = 7, near x = 1, 17, 34, 50, 67, 83, and 100. However, positions intermediate between these charges should represent the maximum coulombic repulsion, so this is not a specific driving force for fragmentation, which is instead consistent with charge site (CAD) or radical site (ECD) initiation. These conclusions were confirmed by studies of a variety of other poly(alkene glycol) polymers. For these, the ECD spectra of the protonated species are consistent with the predicted charge solvation by the ion’s oxygen atoms.

Search for charge-remote reactions of even-electron organic negative ions in the gas phase. Anions derived from disubstituted adamantanes

The collision induced decompositions of 3-substituted adamantanecarboxylate anions have been studied with a view to uncover charge-remote fragmentations of the 3-substituent. The 3-substituent is chosen so that it cannot approach the anion site and therefore any fragmentations of that substituent should proceed independently of the charged centre. (i) Charge-remote radical losses are observed from a 3-CH(Et)2 substituent [e.g. Et˙ and ˙CH(Et)2 losses], but the classical Adams–Gross charge remote loss of an ethene plus dihydrogen is not observed. (ii) Charge-remote loss of MeOD is observed from a 3-C(CD3)2(OMe) substituent together with a number of charge-remote radical losses [e.g. Me˙, MeO˙ and ˙C(CD3)2(OMe)]. (iii) The 3-substituent C(CD3)2 (OCHO) undergoes charge-remote loss of HCO2D for both the carboxylate anion and its corresponding cation, a neutral reaction analogous to both the McLafferty rearrangement of radical cations and the Norrish II diradical rearrangement of aliphatic ketones. (iv) The charge-remote radical losses of MeO˙ and ˙CO2Me occur from a 3-CO2Me substituent.

The facile loss of formic acid from an anion system in which the charged and reacting centres cannot interact

Both the 3-[2-(2-formyloxy-1,3-[2H6]propyl)]adamantane carboxyate anion and cation undergo loss of HCO2D from the 3 substituent by processes which occur remote from and uninfluenced by the carboxylate charged centre.