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Energy Dependence of HCD on Peptide Fragmentation: Stepped Collisional Energy Finds the Sweet Spot
An understanding of the process of peptide fragmentation and what parameters are best to obtain the most useful information is important. This is especially true for large-scale proteomics where data collection and data analysis are most often automated, and manual interpretation of spectra is rare...
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| Published in: | Journal of the American Society for Mass Spectrometry 2013-11, Vol.24 (11), p.1690-1699 |
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| creator | Diedrich, Jolene K. Pinto, Antonio F. M. Yates, John R. |
| description | An understanding of the process of peptide fragmentation and what parameters are best to obtain the most useful information is important. This is especially true for large-scale proteomics where data collection and data analysis are most often automated, and manual interpretation of spectra is rare because of the vast amounts of data generated. We show herein that collisional cell peptide fragmentation, in this case higher collisional dissociation (HCD) in the Q Exactive, is significantly affected by the normalized energy applied. Both peptide sequence and energy applied determine what ion fragments are observed. However, by applying a stepped normalized collisional energy scheme and combining ions from low, medium, and high collision energies, we are able to increase the diversity of fragmentation ions generated. Application of stepped collision energy to HEK293T lysate demonstrated a minimal effect on peptide and protein identification in a large-scale proteomics dataset, but improved phospho site localization through increased sequence coverage. Stepped HCD is also beneficial for tandem mass tagged (TMT) experiments, increasing intensity of TMT reporters used for quantitation without adversely effecting peptide identification.
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| doi_str_mv | 10.1007/s13361-013-0709-7 |
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ᅟ</description><subject>Amino Acid Sequence</subject><subject>Analytical Chemistry</subject><subject>Bioinformatics</subject><subject>Biotechnology</subject><subject>Bronchi - cytology</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Chromatography, Liquid - methods</subject><subject>Cystic Fibrosis - metabolism</subject><subject>Data acquisition</subject><subject>Data analysis</subject><subject>Epithelial Cells - chemistry</subject><subject>Focus: Electron Transfer Dissociation: Research Article</subject><subject>Fragmentation</subject><subject>Fragments</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>Mass spectrometry</subject><subject>Organic Chemistry</subject><subject>Peptide Fragments - analysis</subject><subject>Peptides</subject><subject>Peptides - chemistry</subject><subject>Phosphorylation</subject><subject>Proteomics</subject><subject>Proteomics - methods</subject><subject>Stepped</subject><subject>Tandem Mass Spectrometry - instrumentation</subject><subject>Tandem Mass Spectrometry - methods</subject><issn>1044-0305</issn><issn>1879-1123</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><recordid>eNp1kV9rFDEUxYMotlY_gC8S8MWX0Zs_M5n4IMi2a4WCwupzyCR3tlNmkzHJKv32puxaquBTQu7vnntyDyEvGbxlAOpdZkJ0rAEmGlCgG_WInLJe6YYxLh7XO0jZgID2hDzL-QaAVUo9JSdc6E70TJyS4SJg2t7Sc1wweAwOaRzp5eqcxkC_4lImj3Sd7HaHodgyxfCebgouC3q6ivM85fpkZ3qUWU_BZ1qukW5-IRa6WWJ5Tp6Mds744nieke_ri2-ry-bqy6fPq49XjZMKSqOktqOslj043gmGtmdS9KhkK70cufMtONcPSmnv3OAU77gfBu67ViirUZyRDwfdZT_s0LtqONnZLGna2XRrop3M35UwXZtt_GnqJtpWyyrw5iiQ4o895mJ2U3Y4zzZg3GfDpBQSeMf6ir7-B72J-1QXUSndgtaCSagUO1AuxZwTjvdmGJi7BM0hQVMTNHcJGlV7Xj38xX3Hn8gqwA9ArqWwxfRg9H9VfwMMzKZ3</recordid><startdate>20131101</startdate><enddate>20131101</enddate><creator>Diedrich, Jolene K.</creator><creator>Pinto, Antonio F. 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M.</au><au>Yates, John R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Energy Dependence of HCD on Peptide Fragmentation: Stepped Collisional Energy Finds the Sweet Spot</atitle><jtitle>Journal of the American Society for Mass Spectrometry</jtitle><stitle>J. Am. Soc. Mass Spectrom</stitle><addtitle>J Am Soc Mass Spectrom</addtitle><date>2013-11-01</date><risdate>2013</risdate><volume>24</volume><issue>11</issue><spage>1690</spage><epage>1699</epage><pages>1690-1699</pages><issn>1044-0305</issn><eissn>1879-1123</eissn><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><abstract>An understanding of the process of peptide fragmentation and what parameters are best to obtain the most useful information is important. This is especially true for large-scale proteomics where data collection and data analysis are most often automated, and manual interpretation of spectra is rare because of the vast amounts of data generated. We show herein that collisional cell peptide fragmentation, in this case higher collisional dissociation (HCD) in the Q Exactive, is significantly affected by the normalized energy applied. Both peptide sequence and energy applied determine what ion fragments are observed. However, by applying a stepped normalized collisional energy scheme and combining ions from low, medium, and high collision energies, we are able to increase the diversity of fragmentation ions generated. Application of stepped collision energy to HEK293T lysate demonstrated a minimal effect on peptide and protein identification in a large-scale proteomics dataset, but improved phospho site localization through increased sequence coverage. Stepped HCD is also beneficial for tandem mass tagged (TMT) experiments, increasing intensity of TMT reporters used for quantitation without adversely effecting peptide identification.
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| source | American Chemical Society:Jisc Collections:American Chemical Society Read & Publish Agreement 2022-2024 (Reading list) |
| subjects | Amino Acid Sequence Analytical Chemistry Bioinformatics Biotechnology Bronchi - cytology Chemistry Chemistry and Materials Science Chromatography, Liquid - methods Cystic Fibrosis - metabolism Data acquisition Data analysis Epithelial Cells - chemistry Focus: Electron Transfer Dissociation: Research Article Fragmentation Fragments HEK293 Cells Humans Mass spectrometry Organic Chemistry Peptide Fragments - analysis Peptides Peptides - chemistry Phosphorylation Proteomics Proteomics - methods Stepped Tandem Mass Spectrometry - instrumentation Tandem Mass Spectrometry - methods |
| title | Energy Dependence of HCD on Peptide Fragmentation: Stepped Collisional Energy Finds the Sweet Spot |
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