Zeng et al. JChromB 2016.pdf (774.56 kB)
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Prefractionation methods for individual adult fruit fly hemolymph proteomic analysis

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journal contribution
posted on 02.04.2018 by Q. Zeng, V. Avilov, S.A. Shippy
The analysis of blood provides in depth chemical information of physiological states of organisms. Hemolymph (blood) is the fluid in the open circulatory system of Drosophila melanogaster that is the medium for molecules regulating a wide variety of physiological activities and signaling between tissues. Adult Drosophila is typically less than 3 mm in length and, as a consequence, the available volume of hemolymph is usually less than 50 nL from individual flies. Proteomic analysis of volume-limited hemolymph is a great challenge for both sample handling and subsequent mass spectrometry characterization of this chemically diverse biological fluid with a wide dynamic range of proteins in concentrations. Less abundant proteins, in particular, could be easily lost during sample preparation or missed by current mass spectrometry methods. This article describes simple and customized RPLC column and IEX columns to prefractionate volume-limited hemolymph without excessive dilution. Step-gradient elution methods were developed and optimized to enhance the identification of novel proteins from an individual fruit fly hemolymph sample. Fractions from each step gradient was analyzed by an Agilent nano-RPLC chip column and then characterized by high mass resolution and high mass accuracy orbitrap mass spectrometry. As a result, both RPLC (11 proteins) and IEX fractionation approaches (9 proteins) identified more proteins than an unfractionated control approach with higher protein scores, emPAI values and coverage. Furthermore, a significant number of novel proteins were revealed by both RPLC and IEX fractionation methods, which were missed by unfractionated controls. The demonstration of this method establishes a means to deepen proteomic analysis to this commonly used, important biological model system.

Funding

This work was supported in part by a UIC Provost Award for Graduate Research.

History

Publisher Statement

NOTICE: This is the author’s version of a work that was accepted for publication in Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences. DOI: 10.1016/j.jchromb.2016.02.014

Publisher

Elsevier

Language

en_US

issn

1570-0232

Issue date

01/03/2016

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