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.

A Bruker 7 T Fourier transform (FT) mass spectrometer fitted with electron impact (EI) and electrospray ionization (ESI) external ion sources has been modified for surface-induced dissociation (SID) studies utilizing a self-assembled monolayer (SAM) of CF 3 (CF 2) 9 C 2 H 4 SH alkanethiolate chemisorbed on gold. Operational aspects of ion sources, intermediate hexapole ion storage, timeof-flight function of the ion optics and cell trapping mechanisms are discussed for both primary and secondary ions. Different methods of ion trapping in the FT ion cyclotron resonance cell were analyzed theoretically and dynamic voltage trapping (DVT) was chosen as the most suitable. Direct measurements of primary (short time scale) and secondary (delayed) SID fragmentation and kinetic energy distributions of the SID fragments illustrate the capabilities of the instrument as a research tool for investigating SID kinetics. SID of small molecules and model peptides was investigated over a moderate collision energy range and the ion population was sampled at various decomposition delay times. Kinetic energy distributions of SID fragment ions were measured and shown to be quite low over the range of energies investigated. Most of the primary ion kinetic energy is dissipated into surface excitation and internal energy of ions recoiled from the surface.

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 fragmentsimmonium 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. R esonant harmonic excitation of ions is the primary method for MS detection and activation of induced dissociation in most ion traps. Simultaneous excitation of ions of different m/z ratios is routinely employed to illuminate dissociation pathways using ion traps. It can also be used in harmonic ion guides in order to selectively eject or dissociate ions on their way to an MS analyzer. This paper reports results of validation of a nontrapping multiple frequency component low-energy resonant collision-induced dissociation (CID) in a linear quadrupole ion guide.Several of the ion activation methods used in modern MS instruments are selective, by nature, and target exclusively the parent ion, supplying no additional energy to its primary fragments. As examples of such methods, the sustained off-resonance irradiation (SORI) [1], or on-resonance CID in ion cyclotron resonance (ICR) cells [2] can be mentioned. Other activation methods, such as infrared multi-photon dissociation (IRMPD), and its variations [3,4], or black body infrared dissociation (BIRD) [5] are nonselective, by nature, and always excite primary fragments along with the parent ion. A vast majority of ion activation methods may excite primary fragments depending on the condition of the experiment. In such methods, modeling of the dissociation process can be quite complex. For example, in surface induced dissociation (SID) experiments with moderate impact energies, the dissociation event most likely comprises activation of only the parent ion at the surface, followed by its recoil and subsequent unimolecular decomposition. In contrast, at high impact energies, more extensive fragmentation can be observed due to on-surface shattering of the precursor and possibly some of its primary fragments [6].Axial CID in ion guides (or in triple-quadrupoles) achieved by accelerating the primary ions into the collision cell operated at an elevated pressure is a potentially nonselective activation technique. If initial collisions of the projectile ion with the backgr...

Capillary liquid chromatography (LC) separation coupled with external accumulation Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has recently been demonstrated to have significant potential for proteomics research. Accumulation of an excessive space charge external to the FTICR cell ion trap has been shown to result in increased mass measurement error, undesirable ion discrimination and/or fragmentation, potentially causing misrepresentation or incorrect assignments of lower abundance peptides in the acquired mass spectra. In this work we report on the capability of data-dependent adjustment of ion accumulation times in the course of LC separations, further referred to as automated gain control (AGC). Three different AGC approaches were evaluated based on the number of putative peptides from a tryptic digest of four casein proteins detected in the course of LC/FTICR separations. When compared with the conventional technique, AGC was found to increase, up to a factor of 3, the total number of peptides identified.

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Energy transfer in collisions of benzene molecular ions with a fluorinated self-assembled monolayer surface at normal incidence was studied over the range of collision energies from 0 to 75 eV in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Rice-Ramsperger-Kassel-Marcus (RRKM) theory was used to calculate breakdown graphs for a simplified decomposition scheme of benzene on the time scale of ICR MS detection. Statistical partitioning of excess internal energy between the neutral and the ionic products was included in the theoretical model. Internal energy distributions of the predissociating parent ions were iteratively calculated using the recursive internal energy distribution search (RIEDS) method for each collision energy. On the basis of the above measurements, the collision-energy-dependent E SID f E int energy conversion efficiency was found to maximize at about 19.5% under the conditions of 31 eV SID collision energy.

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