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Comprehensive comparison of collision induced dissociation and electron transfer dissociation.

Molina H, Matthiesen R, Kandasamy K, Pandey A - Anal. Chem. (2008)

Bottom Line: Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique which has proven to be an excellent tool for the elucidation of labile post-translational modifications such as phosphorylation and O-GlcNAcylation of serine and threonine residues.Analysis of approximately 19,000 peptides derived from both standard proteins and complex protein samples revealed that (i) CID identified 50% more peptides than ETD; (ii) ETD resulted in approximately 20% increase in amino acid sequence coverage over CID; and (iii) combining CID and ETD fragmentation increased the sequence coverage for an average tryptic peptide to 92%.Interestingly, our analysis revealed that nearly 60% of all ETD-identified peptides carried two positive charges, which is in sharp contrast to what has been generally accepted.

View Article: PubMed Central - PubMed

Affiliation: McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, The Johns Hopkins University, Baltimore, Maryland 21205, USA.

ABSTRACT
Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique which has proven to be an excellent tool for the elucidation of labile post-translational modifications such as phosphorylation and O-GlcNAcylation of serine and threonine residues. However, unlike collision induced dissociation (CID), which has been studied for decades, the intricacies of ETD-based fragmentation have not yet been firmly established or systematically addressed. In this analysis, we have systematically compared the CID and ETD fragmentation patterns for the large majority of the peptides that do not contain such labile modifications. Using a standard 48 protein mix, we were able to measure false-positive rates for the experiments and also assess a large number of peptides for a detailed comparison of CID and ETD fragmentation pattern. Analysis of approximately 19,000 peptides derived from both standard proteins and complex protein samples revealed that (i) CID identified 50% more peptides than ETD; (ii) ETD resulted in approximately 20% increase in amino acid sequence coverage over CID; and (iii) combining CID and ETD fragmentation increased the sequence coverage for an average tryptic peptide to 92%. Interestingly, our analysis revealed that nearly 60% of all ETD-identified peptides carried two positive charges, which is in sharp contrast to what has been generally accepted. We also present a novel strategy for automatic validation of peptide assignments based on identification of a peptide by consecutive CID and ETD fragmentation in an alternating mode.

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Analysis of a large alternating CID/ETD data set. The CID data were validated using default Spectrum Mill validation criteria whereas the ETD data were validated using a threshold specific for ETD. (A) Peptide length distribution of CID and ETD identified peptides, (B) the distribution of percent sequence coverage for CID and ETD identified peptides. ETD distribution is marked in blue, CID in orange. (C and D) Charge state distributions for CID (C) and ETD (D) identified peptides. (E and F) Scatter plots of sequence coverage for CID (E) and ETD (F) identified peptides as a function for mass-to-charge ratio. Color code: red, 2+ peptides; violet, 3+ peptides; and yellow, 4+ peptides. 
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fig2: Analysis of a large alternating CID/ETD data set. The CID data were validated using default Spectrum Mill validation criteria whereas the ETD data were validated using a threshold specific for ETD. (A) Peptide length distribution of CID and ETD identified peptides, (B) the distribution of percent sequence coverage for CID and ETD identified peptides. ETD distribution is marked in blue, CID in orange. (C and D) Charge state distributions for CID (C) and ETD (D) identified peptides. (E and F) Scatter plots of sequence coverage for CID (E) and ETD (F) identified peptides as a function for mass-to-charge ratio. Color code: red, 2+ peptides; violet, 3+ peptides; and yellow, 4+ peptides. 

Mentions: An analysis of physical properties of the identified peptides revealed that the CID and ETD peptides were very similar with regard to size. On average, a CID identified peptide was composed of 14.4 amino acids where the number was 14.5 for an average ETD peptide. However, as seen from the amino acid length distribution of the CID and ETD peptides (Figure 2A), there is a small trend toward ETD being favorable for peptides containing >13 amino acids. The data, that were searched allowing for two, three, and four positive charges, showed that 57% of the ETD identified peptides carried two positive charges compared to 65% for the CID data set. For triply charged peptides, the numbers for the CID and ETD identifications were 28% and 39%, respectively (parts C and D of Figure 2). The ETD charge state distribution was consistent with our results from the analysis of the standard protein mix (see Figure 1A).


Comprehensive comparison of collision induced dissociation and electron transfer dissociation.

Molina H, Matthiesen R, Kandasamy K, Pandey A - Anal. Chem. (2008)

Analysis of a large alternating CID/ETD data set. The CID data were validated using default Spectrum Mill validation criteria whereas the ETD data were validated using a threshold specific for ETD. (A) Peptide length distribution of CID and ETD identified peptides, (B) the distribution of percent sequence coverage for CID and ETD identified peptides. ETD distribution is marked in blue, CID in orange. (C and D) Charge state distributions for CID (C) and ETD (D) identified peptides. (E and F) Scatter plots of sequence coverage for CID (E) and ETD (F) identified peptides as a function for mass-to-charge ratio. Color code: red, 2+ peptides; violet, 3+ peptides; and yellow, 4+ peptides. 
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664833&req=5

fig2: Analysis of a large alternating CID/ETD data set. The CID data were validated using default Spectrum Mill validation criteria whereas the ETD data were validated using a threshold specific for ETD. (A) Peptide length distribution of CID and ETD identified peptides, (B) the distribution of percent sequence coverage for CID and ETD identified peptides. ETD distribution is marked in blue, CID in orange. (C and D) Charge state distributions for CID (C) and ETD (D) identified peptides. (E and F) Scatter plots of sequence coverage for CID (E) and ETD (F) identified peptides as a function for mass-to-charge ratio. Color code: red, 2+ peptides; violet, 3+ peptides; and yellow, 4+ peptides. 
Mentions: An analysis of physical properties of the identified peptides revealed that the CID and ETD peptides were very similar with regard to size. On average, a CID identified peptide was composed of 14.4 amino acids where the number was 14.5 for an average ETD peptide. However, as seen from the amino acid length distribution of the CID and ETD peptides (Figure 2A), there is a small trend toward ETD being favorable for peptides containing >13 amino acids. The data, that were searched allowing for two, three, and four positive charges, showed that 57% of the ETD identified peptides carried two positive charges compared to 65% for the CID data set. For triply charged peptides, the numbers for the CID and ETD identifications were 28% and 39%, respectively (parts C and D of Figure 2). The ETD charge state distribution was consistent with our results from the analysis of the standard protein mix (see Figure 1A).

Bottom Line: Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique which has proven to be an excellent tool for the elucidation of labile post-translational modifications such as phosphorylation and O-GlcNAcylation of serine and threonine residues.Analysis of approximately 19,000 peptides derived from both standard proteins and complex protein samples revealed that (i) CID identified 50% more peptides than ETD; (ii) ETD resulted in approximately 20% increase in amino acid sequence coverage over CID; and (iii) combining CID and ETD fragmentation increased the sequence coverage for an average tryptic peptide to 92%.Interestingly, our analysis revealed that nearly 60% of all ETD-identified peptides carried two positive charges, which is in sharp contrast to what has been generally accepted.

View Article: PubMed Central - PubMed

Affiliation: McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, The Johns Hopkins University, Baltimore, Maryland 21205, USA.

ABSTRACT
Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique which has proven to be an excellent tool for the elucidation of labile post-translational modifications such as phosphorylation and O-GlcNAcylation of serine and threonine residues. However, unlike collision induced dissociation (CID), which has been studied for decades, the intricacies of ETD-based fragmentation have not yet been firmly established or systematically addressed. In this analysis, we have systematically compared the CID and ETD fragmentation patterns for the large majority of the peptides that do not contain such labile modifications. Using a standard 48 protein mix, we were able to measure false-positive rates for the experiments and also assess a large number of peptides for a detailed comparison of CID and ETD fragmentation pattern. Analysis of approximately 19,000 peptides derived from both standard proteins and complex protein samples revealed that (i) CID identified 50% more peptides than ETD; (ii) ETD resulted in approximately 20% increase in amino acid sequence coverage over CID; and (iii) combining CID and ETD fragmentation increased the sequence coverage for an average tryptic peptide to 92%. Interestingly, our analysis revealed that nearly 60% of all ETD-identified peptides carried two positive charges, which is in sharp contrast to what has been generally accepted. We also present a novel strategy for automatic validation of peptide assignments based on identification of a peptide by consecutive CID and ETD fragmentation in an alternating mode.

Show MeSH
Related in: MedlinePlus