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Liquid chromatography electron capture dissociation tandem mass spectrometry (LC-ECD-MS/MS) versus liquid chromatography collision-induced dissociation tandem mass spectrometry (LC-CID-MS/MS) for the identification of proteins.

Creese AJ, Cooper HJ - J. Am. Soc. Mass Spectrom. (2007)

Bottom Line: Experiments were performed on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer.Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin, lysozyme, cytochrome c, alcohol dehydrogenase, and beta-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides.The results show that although protein coverage was lower for LC-ECD-MS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags, providing greater confidence in protein assignment.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT
Electron capture dissociation (ECD) offers many advantages over the more traditional fragmentation techniques for the analysis of peptides and proteins, although the question remains: How suitable is ECD for incorporation within proteomic strategies for the identification of proteins? Here, we compare LC-ECD-MS/MS and LC-CID-MS/MS as techniques for the identification of proteins. Experiments were performed on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin, lysozyme, cytochrome c, alcohol dehydrogenase, and beta-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides. The results show that although protein coverage was lower for LC-ECD-MS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags, providing greater confidence in protein assignment.

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Total ion current (TIC) chromatogram obtained from an LC-CID-MS/MS (a) and LC-ECD-MS/MS (b) analysis of a tryptic digest of the six-protein mix.
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fig1: Total ion current (TIC) chromatogram obtained from an LC-CID-MS/MS (a) and LC-ECD-MS/MS (b) analysis of a tryptic digest of the six-protein mix.

Mentions: Figure 1 shows the total ion current (TIC) chromatograms for an LC-ECD-MS/MS and an LC-CID-MS/MS analysis. In comparing the two techniques, we applied a practical approach in which the methods are individually optimized such that the maximum number of peptides producing interpretable MS/MS spectra were analyzed. Consequently, experimental parameters are not like for like. CID was performed in the ion trap at a rate of three MS/MS scans/s (single microscan, maximum fill time of 350 ms). ECD was performed in the ICR cell and the resulting mass spectra were the combination of four microscans with a maximum fill time of 4 s (up to 16 s per scan). Therefore over a 60-min gradient, in which peptides are eluting for about 15 min, CID can analyze over 1000 more peptides, leading to greater sequence coverage. One of the major disadvantages of the relatively long analysis time for ECD is that multiply charged peptides of lower intensity could elute without being analyzed.


Liquid chromatography electron capture dissociation tandem mass spectrometry (LC-ECD-MS/MS) versus liquid chromatography collision-induced dissociation tandem mass spectrometry (LC-CID-MS/MS) for the identification of proteins.

Creese AJ, Cooper HJ - J. Am. Soc. Mass Spectrom. (2007)

Total ion current (TIC) chromatogram obtained from an LC-CID-MS/MS (a) and LC-ECD-MS/MS (b) analysis of a tryptic digest of the six-protein mix.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572008&req=5

fig1: Total ion current (TIC) chromatogram obtained from an LC-CID-MS/MS (a) and LC-ECD-MS/MS (b) analysis of a tryptic digest of the six-protein mix.
Mentions: Figure 1 shows the total ion current (TIC) chromatograms for an LC-ECD-MS/MS and an LC-CID-MS/MS analysis. In comparing the two techniques, we applied a practical approach in which the methods are individually optimized such that the maximum number of peptides producing interpretable MS/MS spectra were analyzed. Consequently, experimental parameters are not like for like. CID was performed in the ion trap at a rate of three MS/MS scans/s (single microscan, maximum fill time of 350 ms). ECD was performed in the ICR cell and the resulting mass spectra were the combination of four microscans with a maximum fill time of 4 s (up to 16 s per scan). Therefore over a 60-min gradient, in which peptides are eluting for about 15 min, CID can analyze over 1000 more peptides, leading to greater sequence coverage. One of the major disadvantages of the relatively long analysis time for ECD is that multiply charged peptides of lower intensity could elute without being analyzed.

Bottom Line: Experiments were performed on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer.Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin, lysozyme, cytochrome c, alcohol dehydrogenase, and beta-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides.The results show that although protein coverage was lower for LC-ECD-MS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags, providing greater confidence in protein assignment.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT
Electron capture dissociation (ECD) offers many advantages over the more traditional fragmentation techniques for the analysis of peptides and proteins, although the question remains: How suitable is ECD for incorporation within proteomic strategies for the identification of proteins? Here, we compare LC-ECD-MS/MS and LC-CID-MS/MS as techniques for the identification of proteins. Experiments were performed on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin, lysozyme, cytochrome c, alcohol dehydrogenase, and beta-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides. The results show that although protein coverage was lower for LC-ECD-MS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags, providing greater confidence in protein assignment.

Show MeSH
Related in: MedlinePlus