Anal. Chem. 2006, 78, 6391-6397
Tandem Parallel Fragmentation of Peptides for Mass Spectrometry Alexis A. Ramos,† Hua Yang,† Lauren E. Rosen, and Xudong Yao*
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269
Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collisioninduced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-offlight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.
charged peptide ions are typically selected as precursor ions because they tend to yield significantly more sequence information compared to the corresponding singly charged molecular ion.2,3 Coulombic repulsion is one of the mechanisms thought to promote fragmentation of multiply charged peptide ions.4 Collision-induced fragmentation of ions is generally a function of the collision cross section, collision frequency, ion energy, and charge states.4,5 The mobile proton mechanism provides another model to understand peptide fragmentation.6 Fragmentation pathways of peptide ions in the collision cell are highly dependent on collision energy (CE). Low-energy CID, i.e.,
