Increasing peptide sequence coverage by tandem mass spectrometry improves confidence in database search-based peptide identification and facilitates mapping of post-translational modifications and de novo sequencing. Inducing 2-fold fragmentation by combining electron-transfer and higher-energy collision dissociation (EThcD) generates dual fragment ion series and facilitates extensive peptide backbone fragmentation. After an initial electron-transfer dissociation step, all ions including the unreacted precursor ions are subjected to collision induced dissociation which yields b/y- and c/z-type fragment ions in a single spectrum. This new fragmentation scheme provides richer spectra and substantially increases the peptide sequence coverage and confidence in peptide identification.

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm—incorporating phase information—further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s—increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

Over the past decade peptide sequencing by collision induced dissociation (CID) has become the method of choice in mass spectrometry-based proteomics. The development of alternative fragmentation techniques such as electron transfer dissociation (ETD) has extended the possibilities within tandem mass spectrometry. Recent advances in instrumentation allow peptide fragment ions to be detected with high speed and sensitivity (e.g., in a 2D or 3D ion trap) or at high resolution and high mass accuracy (e.g., an Orbitrap or a ToF). Here, we describe a comprehensive experimental comparison of using ETD, ion-trap CID, and beam type CID (HCD) in combination with either linear ion trap or Orbitrap readout for the large-scale analysis of tryptic peptides. We investigate which combination of fragmentation technique and mass analyzer provides the best performance for the analysis of distinct peptide populations such as N-acetylated, phosphorylated, and tryptic peptides with up to two missed cleavages. We found that HCD provides more peptide identifications than CID and ETD for doubly charged peptides. In terms of Mascot score, ETD FT outperforms the other techniques for peptides with charge states higher than 2. Our data shows that there is a trade-off between spectral quality and speed when using the Orbitrap for fragment ion detection. We conclude that a decision-tree regulated combination of higher-energy collisional dissociation (HCD) and ETD can improve the average Mascot score.

In this work we present the results of a combined experimental and theoretical study concerned with the question how a proton changes the electronic spectrum and dynamics of adenine. In the experimental part, isolated adenine ions have been formed by electro-spray ionisation, stored, mass-selected and cooled in a Paul trap and dissociated by resonant photoexcitation with ns UV laser pulses. The S(0)-S1 spectrum of protonated adenine recorded by fragment ion detection lies in a similar energy range as the first pipi* transition of neutral 9H-adenine. It shows a flat onset with a broad substructure, indicating a large S(0)-S1 geometry shift and an ultra-short lifetime. In the theoretical part, relative energies of the ground and the excited states of the most important tautomers have been calculated by means of a combined density functional theory and multi-reference configuration interaction approach. Protonation at the nitrogen in position 1 of the neutral 9H-adenine tautomer yields the most stable protonated adenine species, 1H-9H-A+. The 3H-7H-A+ and the 3H-9H-A+ tautomers, formed by protonation of 7H- and 9H-adenine in 3-position, are higher in energy by 162 cm(-1) and 688 cm(-1), respectively. Other tautomers lie at considerably higher energies. Calculated vertical absorption spectra are reported for all investigated tautomers whereas geometry optimisations of excited states have been carried out only for the most interesting ones. The S1 state energies and geometries are found to depend on the protonation site. The theoretical data match best with the experimental onset of the spectrum for the 1H-9H-A+ tautomer although we cannot definitely exclude contributions to the experimental spectrum from the 3H-7H-A+ tautomer at higher energies. The vertical S(0)--> S1 excitation energy is similar to the one in neutral 9H-adenine. As for the neutral adenine, we find a conical intersection of the S1 of protonated adenine with the ground state in an out-of-plane coordinate but at lower energies and accessible without barrier.

Hydration of neutral and cationic imidazole is studied by means of ab initio and molecular dynamics calculations, and by photoelectron spectroscopy of the neutral species in a liquid microjet. The calculations show the importance of long range solvent polarization and of the difference between the structure of water molecules in the first shell around the neutral vs cationic species for determining vertical and adiabatic ionization potentials. The vertical ionization potential of neutral imidazole of 8.06 eV calculated using a nonequilibrium polarizable continuum model agrees well with the value of 8.26 eV obtained experimentally for an aqueous solution at pH 10.6.