Infrared multiphoton dissociation (IRMPD) and collisionally activated dissociationof peptides in a quadrupole ion trapwith selective IRMPD of phosphopeptides

Abstract

Dissociation of protonated peptides via infrared multiphoton dissociation (IRMPD) provides more extensive sequence information than is obtained with collisionally activated dissociation (CAD) in a quadrupole ion trap due to the lack of the CAD low m/z cutoff and the ability to form secondary and higher order fragments with the non-resonant photoactivation technique. In addition, IRMPD is shown to be useful for the selective dissociation of phosphopeptides over those which are not phosphorylated because the greater photon absorption efficiency of the phosphorylated peptides leads to their more rapid dissociation. Finally, the selectivity of the IRMPD technique for phosphorylated species in complex mixtures is confirmed with the analysis of a mock peptide mixture and a tryptic digest of ␣-casein. I nvestigations into the protein complement of an organism's genome seek to identify the primary structure of the proteins produced including any modifications that take place. Following transcription, the primary sequence of a protein can undergo modifications such as N-terminal acetylation, formation of disulfide bonds, sulfation, and phosphorylation, all of which affect activity. Arguably, the most important post-translational modification of proteins is the reversible phosphorylation of serine, threonine, and tyrosine residues [1,2]. This covalent modification has regulatory influence over cellular processes such as metabolism, growth, and reproduction [3][4][5]. Because phosphorylation has such an impact on living systems, it is of great interest to be able to determine when, where, and to what extent this type of modification takes place. For this, suitable analytical techniques must be developed and applied. The relative speed and sensitivity of tandem mass spectrometry make it a powerful tool [6 -30] when compared with more conventional methods of phosphoprotein mapping, namely 32 P phosphate labeling of a protein sample followed by purification, enzymatic digestion, chromatographic separation, and Edman sequencing.Collisional activated dissociation (CAD) typically reveals sites of phosphorylation based on a mass loss associated with the cleavage of a phosphate moiety from a peptide ion (Ϫ80 (HPO 3 ) and/or Ϫ98 (H 3 PO 4 ) in positive ion MS/MS) [9,14]. Precursor ion scanning [14,19], neutral loss scanning [23], and nozzle-skimmer dissociation [9] can determine the presence, absence, and location of peptide phosphorylation based on the unique mass losses associated with phosphorylated peptides. In MALDI-TOF mass spectrometry, postsource decay (PSD) of metastable ions formed in the source region via these characteristic pathways can also be used to determine sites of phosphorylation [17]. When a peptide is phosphorylated at a tyrosine residue as opposed to a serine or threonine residue, however, the unique neutral losses are not necessarily observed upon dissociation due to the greater phosphate-tyrosine binding energy and the existence of fewer ␤-elimination pathways than are present in the cases of serine and thr...

Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

“…Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has shown a great capability for the analysis of peptides and proteins, in particular for the characterization of post-translational modifications [22]. The main advantages of this method are the high resolution together with the multiple ion activation modes available, such as (1) CID in the external collision cell or in the ICR cell through sustained off resonance irradiation (SORI) under CID conditions [23], (2) infrared multiphoton dissociation (IRMPD) [24], and (3) for positive ions, electron-capture dissociation (ECD) [25,26]. The latter has revealed particularly powerful for the structural characterization of peptides and proteins [27], given the ability to cleave the peptide backbone while leaving intact labile side-chain modifications, or without disrupting the noncovalent interactions involved in the three-dimensional structure of proteins [28,29].…”

“…SORI CID is an "almost" resonant activation mode where only the precursor ion is effectively activated at its cyclotron frequency. With IRMPD, both the precursor ion and product ions present in the laser beam path are activated, which tend to enhance consecutive dissociations [24]. Thus, the [(b r )* (y s )] two-peptide product ions can dissociate, yielding the conventional y s or b r product ions.…”

Comparison of infrared multiphoton dissociation and collision-induced dissociation of supercharged peptides in ion traps

“…Furthermore, these lower m/z diagnostic ions are important for de novo sequencing algorithms and can be useful for the rapid determination of modified amino acids (i.e., phosphorylated, acetylated, and methylated residues) [26 -28]. Because all ions are continuously irradiated during the activation period, IRMPD can also promote secondary dissociation of primary product ions and thus lead to formation of a more diverse array of diagnostic ions compared with CID [25,29]. Several methods have been explored to combat the low IRMPD efficiencies of larger peptides, including reducing the pressure and/or raising the temperature of the ion trap [28].…”

Shedding Light on the Frontier of Photodissociation

“…Photon-based activation provides a new avenue for exploration in the context of sequencing biopolymers and pinpointing sites of modifications. Third, photoactivation offers the potential for a high degree of selectivity, not necessarily for bond-specific cleavages but for the selective activation of molecules depending on their available chromophores [27,40,42]. This type of selectivity has only rarely been observed for collisional or electronbased activation methods [65][66][67][68].…”