Establishing low-energy sequential decomposition pathways of leucine enkephalin and its N- and C-terminus fragments using multiple-resonance CID in quadrupolar ion guide.

The simultaneous resonant low-energy excitation of leucine enkephalin and its fragment ions was demonstrated in a collision cell of the multipole-quadrupole time-of-flight instrument. Using low-amplitude multiple-resonance excitation CID, we were able to show the exclusive sequential fragmentation of N- and C-terminus fragments all the way to the final fragments--immonium ions of phenylalanine or tyrosine. In this CID mode the single-channel dissociation of each new generation of fragments followed the lowest energy decomposition pathways observable on the time scale of our experiment. Up to six generations of sequential dissociation were carried out in multiple-resonance CID experiments. The direct qualitative comparison of fragmentation of axial-acceleration versus resonant (radial) CID was performed in the same instrument. In both activation methods, fragmentation patterns suggested complex decomposition mechanisms attributable to dynamic competition between sequential and parallel dissociation channels.

Excitation of ions by high-harmonic frequency components in Paul and Penning traps and ion guides. I. Selective simultaneous dipolar excitation of high charge states with clipped sinusoidal and non-harmonic waveforms in a linear quadrupole ion guide.

This article presents a method for simultaneous excitation of multiple high charge states of a molecular ion in Paul or Penning trap. Using a linear quadrupolar ion guide we validate the method by using a variety of time- domain excitation waveforms with high harmonics of the first charge state's resonant frequency. The proposed way of inducing harmonics is the deliberate distortion of the excitation waveform from its sinusoidal form. In order to facilitate interference of the harmonics, a superposition of two sinusoids different by a frequency factor of two is used. The simplest form of distortion - amplitude restriction - of such waveform produces interference of the harmonics and results in selective excitation of charge states. Multiple protonation states of melittin were used as a model in this study.

Low-energy collision-induced dissociation fragmentation analysis of cysteinyl-modified peptides.

The development of methods to chemically modify and isolate cysteinyl-residue-containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotope-coded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolate-specific biotin group to modify and isolate Cys-containing peptides in conjunction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of Deinococcus radiodurans, the presence of these label-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys residue, and to differentiate identical Cys-peptides labeled with either ICAT-d0 or ICAT-d8.