JIOMICS

JIOMICS | VOL 5 | ISSUE 2 | DECEMBER 2015 | 1-62
JOURNAL OF INTEGRATED OMICS
Journal of Integrated Omics
A METHODOLOGICAL JOURNAL
HTTP://WWW.JIOMICS.COM
Special Issue: Proceeding Abstracts of the 4 th International Congress on Analytical Proteomics (ICAP 2015)
Evaluation of alternative strategies depletion proteins for biomarker
discovery in patients serum with bipolar disorder
J.R. Jesus* 1 , G. S. Pessôa 1 , T. B. Lima 2 , F.C. Gozzo 2 , J. L. C. Martínez 3 , M. A. Z. Arruda 1
1
Spectrometry Sample Preparation and Mechanization Group – GEPAM. Institute of Chemistry. University of Campinas - UNICAMP. 13083-
970. Campinas, SP. Brazil. 2 Dalton Mass Spectrometry Group. Institute of Chemistry. University of Campinas - UNICAMP. 13083-970.
Campinas, SP. Brazil. 3 Bioscope group. Chemistry Department. Faculty of Sciences and Technology. New University of Lisbon. Campus de
Caparica. 2829-516. Caparica. Portugal. *Corresponding author: jemmyson.jesus@iqm.unicamp.br
Available Online: 31 December 2015
Abstract
Purpose: Bipolar Disorder (BD) is a mental disease with unclear connections to other similar disorder that affect hundreds of thousand
of individuals in the world. Serum may be utilized as a rich source of biomarker of disease. However, the large number of proteins in serum
makes the analysis very difficult because high abundant proteins (albumin and Immunoglobulin) tend to mask of those of lower abundance.
Depletion of abundant serum proteins may help in the discovery and detection proteins with potential biomarker of diseases, such as bipolar
disorder. Thus, this study had aim evaluate alternative procedure depletion abundant proteins to biomarkers discovery in patient serum samples
with bipolar disorder.
Experimental description: A variety of depletion methods for removal of high abundant proteins from serum have been related in the
literature. This work shows the potential of chemical protein depletion, using dithiothreitol (DTT), acetonitrile (ACN), and tandem depletion
(DTT/ACN). For protein depletion with DTT, approximately 2.0 μL of DTT (500mM) was added to 20 μL (> 10μg μL -1 ) of serum human. Following,
the mixture was incubated for 60 min at 37 °C until a white precipite come to light, which is pellet by centrifugation at 13,000 rpm for
40 min. The supernatant was transferred into microtubes and stored at -20 °C until analysis by SDS-PAGE. For protein depletion with ACN,
20 μL (> 10μg μL -1 ) of serum human was diluted to about 60 μL with Milli-Q water. Then, acetonitrile was added drop by drop to a final concentration
of 60% (v/v). Then, samples were vortexed and sonicated in an ultrasonic bath for 20 min. Supernatant was collected after centrifugation
at 13,000 rpm for 20 min, and evaporated to dryness in SpeedVac. For tandem depletion, the sera were subjected to a sequential depletion
protocol involving the combination of the two precipitation steps previous: protein depletion was first performed with DTT and the supernatant
was treated with ACN in order. Following, the depleted protein were analyzed by SDS-PAGE and all proteic bands were, digested
(trypsin) and analyzed by nanoLC-MS/MS.
Results: A total de 17 proteins were identified for DTT depletion. Of these, 9 belonged to the group of the major proteins (APOA1,
IGHG1, A1AT, C4-A, IGKC, CERUM, APOB, HPT, A1AG1). It was found that this method reduce the presence of albumin in serum human
sample. For ACN depletion, it was found that this method reduces the presence of protein above 75 KDa, for example apoliproteins. The number
of proteins identified in the depleted serum by ACN strategies was 16, of which 6 ( HPT, A1AG1, ALBU, CERUM, IGKC, A1AT) belonged
to the group of the major proteins. 4 (A1AG1, A1AT, CERUM, HPT) were the abundant proteins identified using tandem depletion. Thus, in
this way the relative protein in-gel load of other less-abundant proteins was increased.
Conclusions: In this study, we evaluated the application of DTT, ACN and tandem depletion as an alternative procedure of protein major
depletion for the search of disease biomarker in serum and accordingly tandem depletion present as an efficient tool.
Keywords: Bipolar disorder, depletion protein, serum human, SDS-PAGE, nanoLC-MS/MS.
Acknowledgements: FAPESP, CAPES, CNPq, FAEPEX.
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