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Evaluation of data-dependent and -independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites

Bauer, Manuel and Ahrné, Erik and Baron, Anna P. and Glatter, Timo and Fava, Luca L. and Santamaria, Anna and Nigg, Erich A. and Schmidt, Alexander. (2014) Evaluation of data-dependent and -independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites. Journal of proteome research, Vol. 13, H. 12. pp. 5973-5988.

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Official URL: http://edoc.unibas.ch/dok/A6319316

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Abstract

In recent years, directed and, particularly, targeted mass spectrometric workflows have gained momentum as alternative techniques to conventional data-dependent acquisition (DDA) LC-MS/MS approaches. By focusing on specific peptide species, these methods allow hypothesis-driven analysis of selected proteins of interest, and they have been shown to be suited to monitor low-abundance proteins within complex mixtures. Despite their growing popularity, no study has systematically evaluated these various MS strategies in terms of quantification, detection, and identification limits when they are applied to complex samples. Here, we systematically compared the performance of conventional DDA, directed, and various targeted MS approaches on two different instruments, namely, a hybrid linear ion trap-Orbitrap and a triple quadrupole instrument. We assessed the limits of identification, quantification, and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy-labeled reference peptides, respectively, covering 6 orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed similarly in the absence of background proteins; however, when analyzing whole-cell lysates, targeted methods were at least 5-10 times more sensitive than that of the directed or DDA method. In particular, higher stage fragmentation (MS3) of the neutral loss peak using a linear ion trap increased the dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying nine phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from nonenriched pull-down samples.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Cell Biology (Nigg)
05 Faculty of Science > Departement Biozentrum > Services Biozentrum > Proteomics (Schmidt)
UniBasel Contributors:Nigg, Erich A. and Schmidt, Alexander
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:American Chemical Society
ISSN:1535-3893
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:06 Feb 2015 09:58
Deposited On:05 Dec 2014 09:45

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