Limits...
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.

Show MeSH

Related in: MedlinePlus

ECD (a) and CID (b) mass spectra of the doubly protonated ions of tryptic peptide [421–433] LGEYGFQNALIVR from bovine serum albumin (υ denotes harmonic).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2572008&req=5

fig5: ECD (a) and CID (b) mass spectra of the doubly protonated ions of tryptic peptide [421–433] LGEYGFQNALIVR from bovine serum albumin (υ denotes harmonic).

Mentions: Figures 5, 6, and 7 show LC-ECD-MS/MS and LC-CID-MS/MS mass spectra for three different peptides. The ECD mass spectrum for the bovine serum albumin peptide [LGEYGFQNALIVR] [M+2H]2+ ions (Figure 5a) shows 83% peptide coverage. Ten of the 12 N—Cα bonds were cleaved, eight of which were consecutive. This is a reliable sequence tag. Figure 5b shows the CID mass spectrum for the same species, showing 58% peptide coverage with seven of 12 peptide bonds cleaved; however, only four of these are consecutive. Figure 6a shows the ECD mass spectrum of peptide [TYDSYLGDDYVR] [M+2H]2+ ions from apo-transferrin. The ECD mass spectrum gave 100% peptide coverage: eleven of 11 N—Cα bonds were cleaved, producing a complete sequence tag. The CID mass spectrum of the same peptide (Figure 6b) revealed nine of 11 peptide bonds cleaved, leading to 82% peptide coverage. The nine cleavages are consecutive and thus a reliable sequence tag was obtained. The ECD mass spectrum of [YLYEIAR] [M+2H]2+ ions of bovine serum albumin (Figure 7a) revealed six of 6 N—Cα bonds were cleaved, giving 100% peptide coverage. The CID spectrum for the same peptide resulted in five of six peptide bonds being cleaved with 83% peptide coverage.


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)

ECD (a) and CID (b) mass spectra of the doubly protonated ions of tryptic peptide [421–433] LGEYGFQNALIVR from bovine serum albumin (υ denotes harmonic).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: ECD (a) and CID (b) mass spectra of the doubly protonated ions of tryptic peptide [421–433] LGEYGFQNALIVR from bovine serum albumin (υ denotes harmonic).
Mentions: Figures 5, 6, and 7 show LC-ECD-MS/MS and LC-CID-MS/MS mass spectra for three different peptides. The ECD mass spectrum for the bovine serum albumin peptide [LGEYGFQNALIVR] [M+2H]2+ ions (Figure 5a) shows 83% peptide coverage. Ten of the 12 N—Cα bonds were cleaved, eight of which were consecutive. This is a reliable sequence tag. Figure 5b shows the CID mass spectrum for the same species, showing 58% peptide coverage with seven of 12 peptide bonds cleaved; however, only four of these are consecutive. Figure 6a shows the ECD mass spectrum of peptide [TYDSYLGDDYVR] [M+2H]2+ ions from apo-transferrin. The ECD mass spectrum gave 100% peptide coverage: eleven of 11 N—Cα bonds were cleaved, producing a complete sequence tag. The CID mass spectrum of the same peptide (Figure 6b) revealed nine of 11 peptide bonds cleaved, leading to 82% peptide coverage. The nine cleavages are consecutive and thus a reliable sequence tag was obtained. The ECD mass spectrum of [YLYEIAR] [M+2H]2+ ions of bovine serum albumin (Figure 7a) revealed six of 6 N—Cα bonds were cleaved, giving 100% peptide coverage. The CID spectrum for the same peptide resulted in five of six peptide bonds being cleaved with 83% peptide coverage.

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