Benchmarking common quantification strategies for large-scale phosphoproteomics

Benchmarking common quantification strategies for large-scale phosphoproteomics Comprehensive mass spectrometry (MS)-based proteomics is now feasible, but reproducible quantification remains challenging, especially for post-translational modifications such as phosphorylation. Here, we compare the most popular quantification techniques for global phosphoproteomics: label-free quantification (LFQ), stable isotope labeling by amino acids in cell culture (SILAC) and MS2- and MS3-measured tandem mass tags (TMT). In a mixed species comparison with fixed phosphopeptide ratios, we find LFQ and SILAC to be the most accurate techniques. MS2-based TMT yields the highest precision but lowest accuracy due to ratio compression, which MS3-based TMT can partly rescue. However, MS2-based TMT outperforms MS3-based TMT when analyzing phosphoproteome changes in the DNA damage response, since its higher precision and larger identification numbers allow detection of a greater number of significantly regulated phosphopeptides. Finally, we utilize the TMT multiplexing capabilities to develop an algorithm for determining phosphorylation site stoichiometry, showing that such applications benefit from the high accuracy of MS3-based TMT. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Communications Springer Journals

Benchmarking common quantification strategies for large-scale phosphoproteomics

Loading next page...
 
/lp/springer_journal/benchmarking-common-quantification-strategies-for-large-scale-uteTszCpxO
Publisher
Nature Publishing Group UK
Copyright
Copyright © 2018 by The Author(s)
Subject
Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
eISSN
2041-1723
D.O.I.
10.1038/s41467-018-03309-6
Publisher site
See Article on Publisher Site

Abstract

Comprehensive mass spectrometry (MS)-based proteomics is now feasible, but reproducible quantification remains challenging, especially for post-translational modifications such as phosphorylation. Here, we compare the most popular quantification techniques for global phosphoproteomics: label-free quantification (LFQ), stable isotope labeling by amino acids in cell culture (SILAC) and MS2- and MS3-measured tandem mass tags (TMT). In a mixed species comparison with fixed phosphopeptide ratios, we find LFQ and SILAC to be the most accurate techniques. MS2-based TMT yields the highest precision but lowest accuracy due to ratio compression, which MS3-based TMT can partly rescue. However, MS2-based TMT outperforms MS3-based TMT when analyzing phosphoproteome changes in the DNA damage response, since its higher precision and larger identification numbers allow detection of a greater number of significantly regulated phosphopeptides. Finally, we utilize the TMT multiplexing capabilities to develop an algorithm for determining phosphorylation site stoichiometry, showing that such applications benefit from the high accuracy of MS3-based TMT.

Journal

Nature CommunicationsSpringer Journals

Published: Mar 13, 2018

References

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off