Limits...
Hybridizing ultraviolet photodissociation with electron transfer dissociation for intact protein characterization.

Cannon JR, Holden DD, Brodbelt JS - Anal. Chem. (2014)

Bottom Line: Additionally, the method was shown to decrease spectral congestion via fragmentation of multiple (charge-reduced) precursors.This hybrid activation method was facilitated by performing both ETD and UVPD within the higher energy collisional dissociation (HCD) cell of the Orbitrap mass spectrometer, which afforded an increase in the total number of fragment ions in comparison to the analogous MS(3) format in which ETD and UVPD were undertaken in separate segments of the mass spectrometer.The feasibility of the hybrid method for characterization of proteins on a liquid chromatography timescale characterization was demonstrated for intact ribosomal proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Texas at Austin , 1 University Station A5300, Austin, Texas 78712, United States.

ABSTRACT
We report a hybrid fragmentation method involving electron transfer dissociation (ETD) combined with ultraviolet photodissociation (UVPD) at 193 nm for analysis of intact proteins in an Orbitrap mass spectrometer. Integrating the two fragmentation methods resulted in an increase in the number of identified c- and z-type ions observed when compared to UVPD or ETD alone, as well as generating a more balanced distribution of a/x, b/y, and c/z ion types. Additionally, the method was shown to decrease spectral congestion via fragmentation of multiple (charge-reduced) precursors. This hybrid activation method was facilitated by performing both ETD and UVPD within the higher energy collisional dissociation (HCD) cell of the Orbitrap mass spectrometer, which afforded an increase in the total number of fragment ions in comparison to the analogous MS(3) format in which ETD and UVPD were undertaken in separate segments of the mass spectrometer. The feasibility of the hybrid method for characterization of proteins on a liquid chromatography timescale characterization was demonstrated for intact ribosomal proteins.

Show MeSH

Related in: MedlinePlus

(A) UVPD (one 2.5 mJ laser pulse) of ubiquitin12+, (B) ETUVPD(8 ms ETD in LIT of ubiquitin 13+ followed by one 1.8 mJ laser pulseof ubiquitin 12+·) (MS3), (C) ETUVPD (15 ms ETD of 13+ ubiquitinin HCD cell followed by one 2.5 mJ laser pulse of all product ions).All spectra are shown on the same scale.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4222623&req=5

fig3: (A) UVPD (one 2.5 mJ laser pulse) of ubiquitin12+, (B) ETUVPD(8 ms ETD in LIT of ubiquitin 13+ followed by one 1.8 mJ laser pulseof ubiquitin 12+·) (MS3), (C) ETUVPD (15 ms ETD of 13+ ubiquitinin HCD cell followed by one 2.5 mJ laser pulse of all product ions).All spectra are shown on the same scale.

Mentions: This strategy of simultaneousUVPD of a broader range of precursorsand product ions was implemented and evaluated via ETD of the z = 13 charge state of ubiquitin in the HCD cell followedby UVPD of the entire population of both charge-reduced and nonreducedprecursors as well as product ions arising from ETD. This “broadband”ion activation by UVPD offers two potential benefits. First, the totalion population available for UVPD activation is increased relativeto UVPD of a single charge-reduced species. Second, the potentialfor broad ion isolation in the HCD cell allows detection of a wider m/z range of product ions generated inthe initial ETD reaction as well as the additional ones from UVPD.In the context of characterization of intact proteins, obtaining highsequence coverages and maximizing dissociation efficiencies are premiumbenefits, both of which are feasible with the broadband ETUVPD approach.To capitalize on these benefits, ETUVPD with broad ion isolation wasundertaken for ubiquitin along with comparison to UVPD alone and ETUVPDwith selected ion isolation (see Figure 3).Electron transfer activation in the HCD cell followed by a single5 ns UV pulse (2.5 mJ) resulted in a fragment ion distribution thatresulted from contributions from both ETD and UVPD (Figure 3C). Figure 3 shows expansionsof the spectral region from m/z 720to 780 for UVPD (12+), ETUVPD in which the charge-reduced 12+·ions generated by ETD in the LIT were isolated and subjected to UVPDin the HCD cell, and for ETUVPD in which all the products arisingfrom ETD of the 13+ ions of ubiquitin in the HCD were subsequentlysubjected to UVPD. The shaded regions are unique fragments not seenupon standalone UVPD or UVPD after isolation of the charge-reduced12+· ions in the selective ETD/UVPD spectrum and are only observedupon broadband ETUVPD. Although many of the fragment ions are thesame in all three spectra, the new ones generated upon ETUVPD usingbroad precursor isolation provide additional sequence coverage. Afterdemonstration of feasibility of ETUVPD and evaluation of initial metrics,all subsequent hybrid MS/MS experiments were performed via both ETDand UVPD in the HCD cell.


Hybridizing ultraviolet photodissociation with electron transfer dissociation for intact protein characterization.

Cannon JR, Holden DD, Brodbelt JS - Anal. Chem. (2014)

(A) UVPD (one 2.5 mJ laser pulse) of ubiquitin12+, (B) ETUVPD(8 ms ETD in LIT of ubiquitin 13+ followed by one 1.8 mJ laser pulseof ubiquitin 12+·) (MS3), (C) ETUVPD (15 ms ETD of 13+ ubiquitinin HCD cell followed by one 2.5 mJ laser pulse of all product ions).All spectra are shown on the same scale.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222623&req=5

fig3: (A) UVPD (one 2.5 mJ laser pulse) of ubiquitin12+, (B) ETUVPD(8 ms ETD in LIT of ubiquitin 13+ followed by one 1.8 mJ laser pulseof ubiquitin 12+·) (MS3), (C) ETUVPD (15 ms ETD of 13+ ubiquitinin HCD cell followed by one 2.5 mJ laser pulse of all product ions).All spectra are shown on the same scale.
Mentions: This strategy of simultaneousUVPD of a broader range of precursorsand product ions was implemented and evaluated via ETD of the z = 13 charge state of ubiquitin in the HCD cell followedby UVPD of the entire population of both charge-reduced and nonreducedprecursors as well as product ions arising from ETD. This “broadband”ion activation by UVPD offers two potential benefits. First, the totalion population available for UVPD activation is increased relativeto UVPD of a single charge-reduced species. Second, the potentialfor broad ion isolation in the HCD cell allows detection of a wider m/z range of product ions generated inthe initial ETD reaction as well as the additional ones from UVPD.In the context of characterization of intact proteins, obtaining highsequence coverages and maximizing dissociation efficiencies are premiumbenefits, both of which are feasible with the broadband ETUVPD approach.To capitalize on these benefits, ETUVPD with broad ion isolation wasundertaken for ubiquitin along with comparison to UVPD alone and ETUVPDwith selected ion isolation (see Figure 3).Electron transfer activation in the HCD cell followed by a single5 ns UV pulse (2.5 mJ) resulted in a fragment ion distribution thatresulted from contributions from both ETD and UVPD (Figure 3C). Figure 3 shows expansionsof the spectral region from m/z 720to 780 for UVPD (12+), ETUVPD in which the charge-reduced 12+·ions generated by ETD in the LIT were isolated and subjected to UVPDin the HCD cell, and for ETUVPD in which all the products arisingfrom ETD of the 13+ ions of ubiquitin in the HCD were subsequentlysubjected to UVPD. The shaded regions are unique fragments not seenupon standalone UVPD or UVPD after isolation of the charge-reduced12+· ions in the selective ETD/UVPD spectrum and are only observedupon broadband ETUVPD. Although many of the fragment ions are thesame in all three spectra, the new ones generated upon ETUVPD usingbroad precursor isolation provide additional sequence coverage. Afterdemonstration of feasibility of ETUVPD and evaluation of initial metrics,all subsequent hybrid MS/MS experiments were performed via both ETDand UVPD in the HCD cell.

Bottom Line: Additionally, the method was shown to decrease spectral congestion via fragmentation of multiple (charge-reduced) precursors.This hybrid activation method was facilitated by performing both ETD and UVPD within the higher energy collisional dissociation (HCD) cell of the Orbitrap mass spectrometer, which afforded an increase in the total number of fragment ions in comparison to the analogous MS(3) format in which ETD and UVPD were undertaken in separate segments of the mass spectrometer.The feasibility of the hybrid method for characterization of proteins on a liquid chromatography timescale characterization was demonstrated for intact ribosomal proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Texas at Austin , 1 University Station A5300, Austin, Texas 78712, United States.

ABSTRACT
We report a hybrid fragmentation method involving electron transfer dissociation (ETD) combined with ultraviolet photodissociation (UVPD) at 193 nm for analysis of intact proteins in an Orbitrap mass spectrometer. Integrating the two fragmentation methods resulted in an increase in the number of identified c- and z-type ions observed when compared to UVPD or ETD alone, as well as generating a more balanced distribution of a/x, b/y, and c/z ion types. Additionally, the method was shown to decrease spectral congestion via fragmentation of multiple (charge-reduced) precursors. This hybrid activation method was facilitated by performing both ETD and UVPD within the higher energy collisional dissociation (HCD) cell of the Orbitrap mass spectrometer, which afforded an increase in the total number of fragment ions in comparison to the analogous MS(3) format in which ETD and UVPD were undertaken in separate segments of the mass spectrometer. The feasibility of the hybrid method for characterization of proteins on a liquid chromatography timescale characterization was demonstrated for intact ribosomal proteins.

Show MeSH
Related in: MedlinePlus