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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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Related in: MedlinePlus

Histogram showing the distribution of peptide length of CID (orange) and ETD (blue) matched peptides. Only peptides for which the sequence coverage differed by 20% or more in-between the two fragmentation techniques are counted. The pie chart inserts show the charge state distribution of these two sets of peptides.
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fig6: Histogram showing the distribution of peptide length of CID (orange) and ETD (blue) matched peptides. Only peptides for which the sequence coverage differed by 20% or more in-between the two fragmentation techniques are counted. The pie chart inserts show the charge state distribution of these two sets of peptides.

Mentions: With a CID spectrum and an ETD spectrum of the exact same precursor, identified as the exact same peptide (conditional consecutive CID/ETD validation), it is possible to compare CID and ETD in the most direct manner. First, we wanted to verify our previous observation: ETD identified peptides scores lower than their corresponding CID peptides. For 2+ peptides, the average difference between the CID and ETD scores was 8.5, compared to 3.0 and 3.2 for 3+ and 4+ peptides, respectively. All average differences were in favor of the CID score and in accordance with what we measured for the standard peptides. Of the 9 446 CID/ETD pairs, 3 781 exhibited sequence coverage differences of ≥20% between the two techniques. As expected, for the majority of those CID/ETD pairs (80%), the ETD spectrum provided a better coverage than CID. Comparing the length of the peptides revealed that ETD resulted in a better sequence coverage when peptides were longer (an average length of 14.6 amino acids versus 12.3 for the CID favored peptides) as shown in Figure 6A. The charge state distribution for the peptides where ETD provided a better sequence coverage than CID were very similar to the overall distribution for all ETD identified peptides. However, for the peptides where CID resulted in higher sequence coverage than ETD, we counted that 90% of those peptides were doubly charged. The two distributions are shown in parts B (CID) and C (ETD) of Figure 6.


Comprehensive comparison of collision induced dissociation and electron transfer dissociation.

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

Histogram showing the distribution of peptide length of CID (orange) and ETD (blue) matched peptides. Only peptides for which the sequence coverage differed by 20% or more in-between the two fragmentation techniques are counted. The pie chart inserts show the charge state distribution of these two sets of peptides.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

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

fig6: Histogram showing the distribution of peptide length of CID (orange) and ETD (blue) matched peptides. Only peptides for which the sequence coverage differed by 20% or more in-between the two fragmentation techniques are counted. The pie chart inserts show the charge state distribution of these two sets of peptides.
Mentions: With a CID spectrum and an ETD spectrum of the exact same precursor, identified as the exact same peptide (conditional consecutive CID/ETD validation), it is possible to compare CID and ETD in the most direct manner. First, we wanted to verify our previous observation: ETD identified peptides scores lower than their corresponding CID peptides. For 2+ peptides, the average difference between the CID and ETD scores was 8.5, compared to 3.0 and 3.2 for 3+ and 4+ peptides, respectively. All average differences were in favor of the CID score and in accordance with what we measured for the standard peptides. Of the 9 446 CID/ETD pairs, 3 781 exhibited sequence coverage differences of ≥20% between the two techniques. As expected, for the majority of those CID/ETD pairs (80%), the ETD spectrum provided a better coverage than CID. Comparing the length of the peptides revealed that ETD resulted in a better sequence coverage when peptides were longer (an average length of 14.6 amino acids versus 12.3 for the CID favored peptides) as shown in Figure 6A. The charge state distribution for the peptides where ETD provided a better sequence coverage than CID were very similar to the overall distribution for all ETD identified peptides. However, for the peptides where CID resulted in higher sequence coverage than ETD, we counted that 90% of those peptides were doubly charged. The two distributions are shown in parts B (CID) and C (ETD) of Figure 6.

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