Issue 10, 2010
From the journal:

Analyst

Gaining efficiency by parallel quantification and identification of iTRAQ-labeled peptides using HCD and decision tree guided CID/ETD on an LTQ Orbitrap†

Nikolai Mischerikow,ab   Pim van Nierop,c   Ka Wan Li,c   Hans-Gert Bernstein,d   August B. Smit,c   Albert J. R. Heck*abe  and  A. F. Maarten Altelaar*ab  

* Corresponding authors

a Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, Utrecht, The Netherlands
E-mail: a.j.r.heck@uu.nl, m.altelaar@uu.nl
Fax: +31 30 2536919
Tel: +31 30 253 9554

b Netherlands Proteomics Centre, Padualaan 8, Utrecht, The Netherlands

c Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, VU University Amsterdam, De Boelelaan 1085, Amsterdam, The Netherlands

d Department of Psychiatry, University of Magdeburg, Leipziger Strasse 44, Magdeburg, Germany

e Centre for Biomedical Genetics, Padualaan 8, Utrecht, The Netherlands

Abstract

Isobaric stable isotope labeling of peptides using iTRAQ is an important method for MS based quantitative proteomics. Traditionally, quantitative analysis of iTRAQ labeled peptides has been confined to beam-type instruments because of the weak detection capabilities of ion traps for low mass ions. Recent technical advances in fragmentation techniques on linear ion traps and the hybrid linear ion trap-orbitrap allow circumventing this limitation. Namely, PQD and HCD facilitate iTRAQ analysis on these instrument types. Here we report a method for iTRAQ-based relative quantification on the ETD enabled LTQ OrbitrapXL, which is based on parallel peptide quantification and peptide identification. iTRAQ reporter ion generation is performed by HCD, while CID and ETD provide peptide identification data in parallel in the LTQ ion trap. This approach circumvents problems accompanying iTRAQ reporter ion generation with ETD and allows quantitative, decision tree-based CID/ETD experiments. Furthermore, the use of HCD solely for iTRAQ reporter ion read out significantly reduces the number of ions needed to obtain informative spectra, which significantly reduces the analysis time. Finally, we show that integration of this method, both with existing CID and ETD methods as well as with existing iTRAQ data analysis workflows, is simple to realize. By applying our approach to the analysis of the synapse proteome from human brain biopsies, we demonstrate that it outperforms a latest generation MALDITOF/TOF instrument, with improvements in both peptide and protein identification and quantification. Conclusively, our work shows how HCD, CID and ETD can be beneficially combined to enable iTRAQ-based quantification on an ETD-enabled LTQOrbitrap XL.

Graphical abstract: Gaining efficiency by parallel quantification and identification of iTRAQ-labeled peptides using HCD and decision tree guided CID/ETD on an LTQ Orbitrap

About
Cited by
Related
Download options Please wait...

Supplementary files

Article information

DOI
https://doi.org/10.1039/C0AN00267D
Article type
Paper
Submitted
26 Apr 2010
Accepted
05 Jul 2010
First published
10 Aug 2010

Analyst, 2010,135, 2643-2652

Permissions

Request permissions

Gaining efficiency by parallel quantification and identification of iTRAQ-labeled peptides using HCD and decision tree guided CID/ETD on an LTQ Orbitrap

N. Mischerikow, P. van Nierop, K. W. Li, H. Bernstein, A. B. Smit, A. J. R. Heck and A. F. M. Altelaar, Analyst, 2010, 135, 2643 DOI: 10.1039/C0AN00267D

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements