Electron capture dissociation (ECD) [1] -an experiment generally performed within the high magnetic field of a Fourier transform ion cyclotron resonance mass spectrometerresults from the mutual storage of thermal electrons with multiply protonated peptide cations. The technique is particularly useful, as it generates random backbone cleavage with little regard to the presence of posttranslational modifications (PTMs), amino acid composition, or peptide length. Electron transfer dissociation (ETD), [2] the ion-ion analogue of ECD, is conducted in radio-frequency (RF) quadrupole ion trap devices, in which radical anions serve as electron donors. Since it can be implemented on virtually any mass spectrometer with an RF ion transfer or storage device, ETD has become an increasingly widespread dissociation method.The capture of an electron can trigger a free-radicaldriven rearrangement that results in N À C a backbone cleavage and the production of c-and zC-type fragment ions. Sometimes, however, the precursor cation captures the electron and forms a long-lived, charge-reduced species that does not separate (an ECnoD or ETnoD product).[3] This phenomenon becomes more probable as the mass-to-charge (m/z) ratio of the precursor increases. As the charge density decreases, the magnitude of intramolecular noncovalent interactions increases, so that the newly formed c-and zC-type fragment ions often remain bound following electron capture and cleavage-an obstacle of higher consequence for ETD, which is conducted under conditions of elevated pressure.[4] McLafferty and co-workers reported that photon bombardment of the precursor cation prior to ECD (activated-ion ECD, AI ECD) decreased nondissociative electron capture, [5] presumably by destroying the secondary structure of the peptide cation prior to electron capture.ETD is conducted at pressures that are approximately 10 6 times higher than those used in ECD (which is carried out at approximately 0.13 Pa). Therefore, precursor cations undergoing ETD are considerably cooler, and preactivation either with photons or through collisions is expected to produce only short-lived (< 1 ms) unfolding. Recently, we examined the use of collisions to coerce the ETnoD products into dissociating through a technique coined ETcaD (ETD in conjunction with collisional activation).[3] The method increased the number and intensity of N À C a backbone cleavages; however, the majority of the newly formed fragment ions displayed evidence of hydrogen-atom rearrangement to produce evenelectron z-type fragments and odd-electron cC-type products. ECD practitioners propose that such rearrangements occur because the c-and zC-type fragment ions are held in close proximity, so that an H atom can be abstracted from the ctype and directed to the zC-type product (this hydrogen-atom transfer occurs prior to the separation of the two fragment ions). [6,7] For large-scale sequencing applications, these rearrangements are problematic, as the mass window needed to define a possible fragment becomes too large.We ...

We have developed a new IR chromogenic cross-linker (IRCX) to aid in rapidly distinguishing cross-linked peptides from unmodified species in complex mixtures. By incorporating a phosphate functional group into the cross-linker, one can take advantage of its unique IR absorption properties, affording selective infrared multiphoton dissociation (IRMPD) of the cross-linked peptides. In a mock mixture of unmodified peptides and IRCX-cross-linked peptides (intramolecularly and intermolecularly cross-linked), only the peptides containing the IRCX modification were shown to dissociate upon exposure to 50 ms of 10.6-microm radiation. LC-IRMPD-MS proved to be an effective method to distinguish the cross-linked peptides in a tryptic digest of IRCX-cross-linked ubiquitin. A total of four intermolecular cross-links and two dead-end modifications were identified using IRCX and LC-IRMPD-MS. IRMPD of these cross-linked peptides resulted in secondary dissociation of all primary fragment ions containing the chromophore, producing a series of unmodified b- or y-type ions that allowed the cross-linked peptides to be sequenced without the need for collision-induced dissociation.

A dual pressure linear ion trap mass spectrometer was modified to permit infrared multiphoton dissociation (IRMPD) in each of the two cells -the first a high pressure cell operated at nominally 5 × 10 -3 Torr and the second a low pressure cell operated at nominally 3 × 10 -4 Torr. When IRMPD was performed in the high pressure cell, most peptide ions did not undergo significant photodissociation; however, in the low pressure cell peptide cations were efficiently dissociated with less than 25 ms of IR irradiation regardless of charge state. IRMPD of peptide cations allowed the detection of low m/z product ions including the y 1 fragments and immonium ions which are not typically observed by ion trap collision induced dissociation (CID). Photodissociation efficiencies of ~100% and MS/MS (tandem mass spectrometry) efficiencies of greater than 60% were observed for both multiply and singly protonated peptides. In general, higher sequence coverage of peptides was obtained using IRMPD over CID. Further, greater than 90% of the product ion current in the IRMPD mass spectra of doubly charged peptide ions was composed of singly charged product ions compared to the CID mass spectra in which the abundances of the multiply and singly charged product ions were equally divided. Highly charged primary product ions also underwent efficient photodissociation to yield singly charged secondary product ions, thus simplifying the IRMPD product ion mass spectra.