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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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Heatmap showing averaged ETD over CID intensity ratios. Green indicates favorable ETD fragmentation over CID, while red indicates a favorable CID over ETD fragmentation. Black indicates differences of less than 2-fold. The heatmap is generated from the spectra of all the >9 000 peptides identified by consecutive CID/ETD. “m” indicates oxidized methionine. All cysteines, C, are alkylated. Similar diagrams, but divided into charge states (2+ and 3+) are shown in Supporting Information Figure 1.
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fig7: Heatmap showing averaged ETD over CID intensity ratios. Green indicates favorable ETD fragmentation over CID, while red indicates a favorable CID over ETD fragmentation. Black indicates differences of less than 2-fold. The heatmap is generated from the spectra of all the >9 000 peptides identified by consecutive CID/ETD. “m” indicates oxidized methionine. All cysteines, C, are alkylated. Similar diagrams, but divided into charge states (2+ and 3+) are shown in Supporting Information Figure 1.

Mentions: To assess the complementary nature of CID and ETD fragmentation, we compared their fragmentation patterns in relation to neighboring amino acids using the 9 446 pairs of spectra obtained in the conditional consecutive CID/ETD validation. This was done by extracting the signals of a-, b-, y-, (y − 17)-, and (y − 18)-ions (CID) and c-, c′-, z-, z′-, and z′′-ions (ETD) for each peptide. A 21 × 21 matrix of average and normalized ion intensities for fragmentation between two amino acids A and B was created. Methionine oxidation and all natural occurring amino acids were considered. The matrices for CID and ETD data are calculated by the following formulas:where x is the position in the sequence of the amino acids A and B. n is the length of the sequence, I is the intensity of the different ion types, and Itotal is the sum of all observed intensity from every ion type generated from all possible positions of fragmentation. Because our analysis is conducted using the exact same peptides subjected to both CID and ETD, we chose to focus on the relative differences between the CID and ETD data sets by using the ratios of FAB,ETD/FAB,CID (eqs 1 and 2). In a heatmap (Figure 7), where all combinations of amino acid pairs are shown, we plotted three states being either (i) no change defined as less than a 2-fold difference between CID and ETD (black), (ii) a 2-fold or greater difference in favor of ETD (red), and (iii) a 2-fold or greater difference in favor of CID (green). From the heatmap, it is clear that ETD fragmentation is more pronounced then CID fragmentation when amino acids are flanked by the basic residues: lysine (K), arginine (R), and histidine (H). In the heatmap, this is shown by red boxes both vertically and horizontally for the amino acids K, R, and H. Similar observation has been made earlier for ECD fragmentation of ubiquitin.(23) A possible explanation is that the positive charged residues facilitate the capture of the transferred electrons. Another striking pattern is the lack of fragmentation in ETD, compared to CID, when proline is C-terminal to any preceding residue (vertical green area for C-terminal proline). This observation has previously been reported for ECD.(24) Though less prominent, the heat map also suggests that ETD fragmentation is less favorable than CID for amino acids flanked by the sulfur containing amino acids methionine and cysteine. It has been shown that the presence of alkylated cysteines and methionines in a peptide sequence lead to dominant side chain fragmentation.(25) Thus, it is likely that the sulfur containing side chain is favored over the adjacent backbone carbonyl site, inhibiting the fragmentation at these sites and leading to these dominant neutral losses.


Comprehensive comparison of collision induced dissociation and electron transfer dissociation.

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

Heatmap showing averaged ETD over CID intensity ratios. Green indicates favorable ETD fragmentation over CID, while red indicates a favorable CID over ETD fragmentation. Black indicates differences of less than 2-fold. The heatmap is generated from the spectra of all the >9 000 peptides identified by consecutive CID/ETD. “m” indicates oxidized methionine. All cysteines, C, are alkylated. Similar diagrams, but divided into charge states (2+ and 3+) are shown in Supporting Information Figure 1.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

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

fig7: Heatmap showing averaged ETD over CID intensity ratios. Green indicates favorable ETD fragmentation over CID, while red indicates a favorable CID over ETD fragmentation. Black indicates differences of less than 2-fold. The heatmap is generated from the spectra of all the >9 000 peptides identified by consecutive CID/ETD. “m” indicates oxidized methionine. All cysteines, C, are alkylated. Similar diagrams, but divided into charge states (2+ and 3+) are shown in Supporting Information Figure 1.
Mentions: To assess the complementary nature of CID and ETD fragmentation, we compared their fragmentation patterns in relation to neighboring amino acids using the 9 446 pairs of spectra obtained in the conditional consecutive CID/ETD validation. This was done by extracting the signals of a-, b-, y-, (y − 17)-, and (y − 18)-ions (CID) and c-, c′-, z-, z′-, and z′′-ions (ETD) for each peptide. A 21 × 21 matrix of average and normalized ion intensities for fragmentation between two amino acids A and B was created. Methionine oxidation and all natural occurring amino acids were considered. The matrices for CID and ETD data are calculated by the following formulas:where x is the position in the sequence of the amino acids A and B. n is the length of the sequence, I is the intensity of the different ion types, and Itotal is the sum of all observed intensity from every ion type generated from all possible positions of fragmentation. Because our analysis is conducted using the exact same peptides subjected to both CID and ETD, we chose to focus on the relative differences between the CID and ETD data sets by using the ratios of FAB,ETD/FAB,CID (eqs 1 and 2). In a heatmap (Figure 7), where all combinations of amino acid pairs are shown, we plotted three states being either (i) no change defined as less than a 2-fold difference between CID and ETD (black), (ii) a 2-fold or greater difference in favor of ETD (red), and (iii) a 2-fold or greater difference in favor of CID (green). From the heatmap, it is clear that ETD fragmentation is more pronounced then CID fragmentation when amino acids are flanked by the basic residues: lysine (K), arginine (R), and histidine (H). In the heatmap, this is shown by red boxes both vertically and horizontally for the amino acids K, R, and H. Similar observation has been made earlier for ECD fragmentation of ubiquitin.(23) A possible explanation is that the positive charged residues facilitate the capture of the transferred electrons. Another striking pattern is the lack of fragmentation in ETD, compared to CID, when proline is C-terminal to any preceding residue (vertical green area for C-terminal proline). This observation has previously been reported for ECD.(24) Though less prominent, the heat map also suggests that ETD fragmentation is less favorable than CID for amino acids flanked by the sulfur containing amino acids methionine and cysteine. It has been shown that the presence of alkylated cysteines and methionines in a peptide sequence lead to dominant side chain fragmentation.(25) Thus, it is likely that the sulfur containing side chain is favored over the adjacent backbone carbonyl site, inhibiting the fragmentation at these sites and leading to these dominant neutral losses.

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