Optimization of plasma sample pretreatment for quantitative analysis using iTRAQ labeling and LC-MALDI-TOF/TOF

Shotgun proteomic methods involving iTRAQ (isobaric tags for relative and absolute quantitation) peptide labeling facilitate quantitative analyses of proteomes and searches for useful biomarkers. However, the plasma proteome's complexity and the highly dynamic plasma protein concentration range...

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Bibliographic Details
Published in:PloS one 2014-07, Vol.9 (7), p.e101694-e101694
Main Authors: Luczak, Magdalena, Marczak, Lukasz, Stobiecki, Maciej
Format: Article
Language:English
Subjects:
Affinity
Biology and Life Sciences
Biomarkers
Blood Proteins - analysis
Blood Proteins - isolation & purification
Cancer
Cardiovascular disease
Chemical Fractionation - methods
Chemistry
Chromatography
Chromatography, Ion Exchange - methods
Comparative analysis
Complexity
Depletion
Fractionation
Humans
Labeling
Labelling
Mass spectrometry
Medical research
Medicine and Health Sciences
Methods
Optimization
Peptides
Physical Sciences
Plasma
Pretreatment
Proteins
Proteomics
Proteomics - methods
Quantitation
Quantitative analysis
Reagents
Reproducibility
Research and Analysis Methods
Sample preparation
Scientific imaging
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
Ultrafiltration
Ultrafiltration - methods
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Summary:Shotgun proteomic methods involving iTRAQ (isobaric tags for relative and absolute quantitation) peptide labeling facilitate quantitative analyses of proteomes and searches for useful biomarkers. However, the plasma proteome's complexity and the highly dynamic plasma protein concentration range limit the ability of conventional approaches to analyze and identify a large number of proteins, including useful biomarkers. The goal of this paper is to elucidate the best approach for plasma sample pretreatment for MS- and iTRAQ-based analyses. Here, we systematically compared four approaches, which include centrifugal ultrafiltration, SCX chromatography with fractionation, affinity depletion, and plasma without fractionation, to reduce plasma sample complexity. We generated an optimized protocol for quantitative protein analysis using iTRAQ reagents and an UltrafleXtreme (Bruker Daltonics) MALDI TOF/TOF mass spectrometer. Moreover, we used a simple, rapid, efficient, but inexpensive sample pretreatment technique that generated an optimal opportunity for biomarker discovery. We discuss the results from the four sample pretreatment approaches and conclude that SCX chromatography without affinity depletion is the best plasma sample preparation pretreatment method for proteome analysis. Using this technique, we identified 1,780 unique proteins, including 1,427 that were quantified by iTRAQ with high reproducibility and accuracy.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0101694