NDLI: Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry

Content Provider	Springer Nature Link
Author	Hsu, Chuan Chih Xue, Liang Arrington, Justine V. Wang, Pengcheng Paez Paez, Juan Sebastian Zhou, Yuan Zhu, Jian Kang Tao, W. Andy
Copyright Year	2017
Abstract	Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutral loss. Phosphoproteomics has become an important dimension in systems biology studies, and it is essential to have efficient analytical tools to cover a broad range of signaling events. To evaluate current mass spectrometric performance, we present here a novel method to estimate the efficiency of phosphopeptide identification by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with one of three purified kinases—casein kinase II, mitogen-activated protein kinase 6, and SNF-related protein kinase 2.6—along with $^{16}$O$_{4}$- and $^{18}$O$_{4}$-ATP separately for in vitro kinase reactions. Phosphopeptides were enriched and analyzed by LC-MS. The phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall, we found that current high speed and high accuracy mass spectrometers can only identify 20%–40% of total phosphopeptides primarily due to relatively poor fragmentation, additional modifications, and low abundance, highlighting the urgent need for continuous efforts to improve phosphopeptide identification efficiency. Graphical Abstract ᅟ
Starting Page	1127
Ending Page	1135
Page Count	9
File Format	PDF
ISSN	10440305
Journal	Journal of The American Society for Mass Spectrometry
Volume Number	28
Issue Number	6
e-ISSN	18791123
Language	English
Publisher	Springer US
Publisher Date	2017-03-10
Publisher Institution	The American Society for Mass Spectrometry
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Protein phosphorylation Proteomics Tandem mass spectrometry Analytical Chemistry Biotechnology Organic Chemistry Bioinformatics
Content Type	Text
Resource Type	Article
Subject	Spectroscopy Structural Biology

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3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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