David M. Horn

Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules

Here a fully automated computer algorithm is applied to complex mass spectra of peptides and proteins. This method uses a subtractive peak finding routine to locate possible isotopic clusters in the spectrum, subjecting these to a combination of the previous Fourier transform/ Patterson method for primary charge determination and the method for least-squares fitting to a theoretically derived isotopic abundance distribution for m/z determination of the most abundant isotopic peak, and the statistical reliability of this determination. If a predicted protein sequence is available, each such m/z value is checked for assignment as a sequence fragment. A new signal-to-noise calculation procedure has been devised for accurate determination of baseline and noise width for spectra with high peak density. In 2 h, the program identified 824 isotopic clusters representing 581 mass values in the spectrum of a GluC digest of a 191 kDa protein; this is \s>50% more than the number of mass values found by the extremely tedious operator-applied methodology used previously. The program should be generally applicable to classes of large molecules, including DNA and polymers. Thorough high resolution analysis of spectra by Horn (THRASH) is proposed as the program’s verb.

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.

ELECTRON CAPTURE DISSOCIATION OF MULTIPLY CHARGED PEPTIDE CATIONS

Abstract For five 12- to 17-mer multiply charged peptide cations, capture of low energy electrons yields unique products, mainly c and z · ions from amine bond cleavage. Their mass values for m > 400 define the complete sequence for two peptides, all but the ordering of a doublet in another, and all but the partial ordering of a triplet in the other two. The mass values from collisionally activated dissociation (CAD), on the other hand, indicate cleavages of 33 amide bonds ( b and y ion products) of the 68 possible bonds between the amino acids of these peptides. Because the other common methods for ion dissociation yield products similar to those from CAD, electron capture dissociation (ECD) should provide a valuable complementary technique for sequencing of multiply charged peptide cations.

Electron capture versus energetic dissociation of protein ions

Abstract The identity of neighboring amino acids has little influence on the dissociation of multiply protonated proteins by electron capture dissociation. As exceptions, no cleavage occurs on the N-terminal side of Pro, and little on either side of Cys, whereas the C-terminal side of Trp is heavily favored. The neighboring amino acids have a far greater effect on energetic dissociation, making the combined methods promising for the de novo sequencing of proteins.

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.

Blackbody infrared radiative dissociation of larger (42 kDa) multiply charged proteins

Abstract Blackbody infrared radiative dissociation (BIRD), demonstrated originally with ions as large as 17 kDa, has been applied to larger proteins in a 6 T Fourier-transform mass spectrometer. For carbonic anhydrase (29 kDa), ThiF (27 kDa), and thiazole kinase (29 kDa), ion cell temperatures of 60–110 °C give mostly uninformative H 2 O loss, but 145 °C gives extensive backbone dissociation. For carbonic anhydrase ions at 70 °C, H 2 O loss continues for >240 s; thermalizing ions for ∼30 s reduces H 2 O loss eightfold. For thiaminase I (42 kDa), H 2 O loss is not observed, with backbone dissociation occurring above 150 °C. For these proteins, BIRD has effected cleavages of 34, 41, 23, and 28, respectively, backbone bonds. Although most are the same as those cleaved by infrared multiphoton dissociation and collisionally activated dissociation, some BIRD cleavages do provide additional and complementary sequence information. Carbonic anhydrase also shows extensive H 2 O loss from its fragment ions that compromises their validity for sequencing.

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.

Electron Capture Dissociation Produces Many More Protein Backbone Cleavages Than Collisional and IR Excitation

Capture of free, low energy ( 10 kDa, and proteins >20 kDa only undergo multiple reduction steps without apparent fragmentation.