Thermally Assisted Infrared Multiphoton Photodissociation in a Quadrupole Ion Trap

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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...

mentioning

confidence: 99%

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

Crowe

Brodbelt

2004

112

“…One potential downside of IRMPD is that activation is less efficient at higher pressures because of the competition from collisional cooling; however, trapping is less effective at lower pressures. A strategy for alleviating this trade-off has been reported recently by the combination of IRMPD with heating of the trap, thus improving energy deposition while maintaining satisfactory trapping efficiency by allowing use of higher pressures [29]. Although not used in the present application because of the naturally high dissociation efficiencies of the erythromycin compounds, heating should offset this drawback of IRMPD for other applications.…”

mentioning

confidence: 99%

Characterization of erythromycin analogs by collisional activated dissociation and infrared multiphoton dissociation in a quadrupole ion trap

Crowe

Brodbelt

Goolsby

et al. 2002

The effectiveness of two activation techniques, collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD), is compared for structural characterization of protonated and lithium-cationized macrolides and a series of synthetic precursors in a quadrupole ion trap (QIT). Generally, cleavage of the glycosidic linkages attaching the sugars to the macrolide ring and water losses constitute the major fragmentation pathways for most of the protonated compounds. In the IRMPD spectra, a diagnostic fragment ion assigned as the desosamine ion is a dominant ion that is not observed in the CAD spectra because of the higher m/z limit of the storage range required during collisional activation. Activation of the lithium-cationized species results in new diagnostic fragmentation pathways that are particularly useful for confirming the identities of the protecting groups in the synthetic precursors. Multi-step IRMPD allows mapping of the fragmentation genealogies in greater detail and supports the proposed structures of the fragment ions. (J Am Soc Mass Spectrom 2002, 13, 630 -649)

“…Sequential dissociation of product ions is increased by focusing the laser and by operating at an increased bath gas pressure to minimize the size of the ion cloud. nfrared multiphoton photodissociation (IRMPD) has been used for tandem mass spectrometry in a quadrupole ion trap mass spectrometer [1][2][3][4][5][6][7] and a Fourier transform ion cyclotron resonance mass spectrometer [8 -11] as an alternative to collision-induced dissociation (CID). Only one m/z ion is selected for excitation in conventional CID, limiting sequential dissociation of product ions into smaller mass product ions.…”

mentioning

confidence: 99%

“…Conversely, in IRMPD all parent and product ions are irradiated simultaneously with a collimated, IR laser providing the capability to generate abundant small mass product ions. IRMPD is typically performed in a quadrupole ion trap mass spectrometer at a smaller low mass cut-off (LMCO) than CID, allowing the observation of a broader m/z range of product ions [2,4], and a higher MS/MS efficiency,where E MS/MS is MS/MS efficiency, F i is the abundance of each product ion, and P 0 is the initial abundance of the parent ion before irradiation. IRMPD is performed in a Fourier transform ion cyclotron resonance mass spectrometer to eliminate the need for a CID target gas to be leaked into and pumped out of the ICR cell [10,11].…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Improving IRMPD in a quadrupole ion trap

Newsome

Glish

2009

Self Cite

A focused laser is used to make infrared multiphoton photodissociation (IRMPD) more efficient in a quadrupole ion trap mass spectrometer. Efficient (up to 100%) dissociation at the standard operating pressure of 1 ϫ 10 Ϫ3 Torr can be achieved without any supplemental ion activation and with shorter irradiation times. The axial amplitudes of trapped ion clouds are measured using laser tomography. Laser flux on the ion cloud is increased six times by focusing the laser so that the beam waist approximates the ion cloud size. Unmodified peptide ions from 200 Da to 3 kDa are completely dissociated in 2.5-10 ms at a bath gas pressure of 3.3 ϫ 10 Ϫ4 Torr and in 3-25 ms at 1.0 ϫ 10 Ϫ3 Torr. Sequential dissociation of product ions is increased by focusing the laser and by operating at an increased bath gas pressure to minimize the size of the ion cloud. nfrared multiphoton photodissociation (IRMPD) has been used for tandem mass spectrometry in a quadrupole ion trap mass spectrometer [1][2][3][4][5][6][7] and a Fourier transform ion cyclotron resonance mass spectrometer [8 -11] as an alternative to collision-induced dissociation (CID). Only one m/z ion is selected for excitation in conventional CID, limiting sequential dissociation of product ions into smaller mass product ions. Conversely, in IRMPD all parent and product ions are irradiated simultaneously with a collimated, IR laser providing the capability to generate abundant small mass product ions. IRMPD is typically performed in a quadrupole ion trap mass spectrometer at a smaller low mass cut-off (LMCO) than CID, allowing the observation of a broader m/z range of product ions [2,4], and a higher MS/MS efficiency,where E MS/MS is MS/MS efficiency, F i is the abundance of each product ion, and P 0 is the initial abundance of the parent ion before irradiation. IRMPD is performed in a Fourier transform ion cyclotron resonance mass spectrometer to eliminate the need for a CID target gas to be leaked into and pumped out of the ICR cell [10,11]. The sensitivity of a quadrupole ion trap mass spectrometer is optimized with a bath gas pressure around 1.0 ϫ 10 Ϫ3 Torr. Bath gas collisions reduce the kinetic energy of trapped ions and minimize the size of the ion cloud [12]. However, collisions also remove internal energy gained from IR absorption, necessitating longer periods of irradiation for dissociation and decreasing fragmentation efficiency [13],where E F is fragmentation efficiency, F i is the abundance of each product ion, and P is the abundance of the parent ion remaining after irradiation. IRMPD in a quadrupole ion trap mass spectrometer often is performed at 1 ϫ 10 Ϫ5 to 3 ϫ 10 Ϫ4 Torr to decrease collisional cooling of parent ion internal energy [2-7], [13] and achieve dissociation with shorter irradiation times. However, this reduced bath gas pressure is less effective for trapping ions during injection and reduces sensitivity.Several methods have been developed to increase IRMPD fragmentation efficiency by combining IRMPD with another form of ion activation an...