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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Bibliographic Details
Published in:Journal of the American Society for Mass Spectrometry 2013-11, Vol.24 (11), p.1690-1699
Main Authors: Diedrich, Jolene K., Pinto, Antonio F. M., Yates, John R.
Format: Article
Language:English
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
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Summary: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. Figure ᅟ
ISSN:1044-0305
1879-1123
DOI:10.1007/s13361-013-0709-7