Issue 7, 2009
From the journal:

Lab on a Chip

When one chip is not enough: Augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens

Greplova Kristina,a   Pilny Radomir,a   Budinska Eva,b   Dubska Lenka,a   Lakomy Radek,c   Vyzula Rostislav,c   Vojtesek Borivojd  and  Valik Dalibor*e  

* Corresponding authors

a Department of Laboratory Medicine, Masaryk Memorial Cancer Institute, Brno, Czech Republic

b Institute of Biostatistics and Analyses, Brno, Czech Republic

c Department of Clinical Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic

d Section of Experimental Pathology, Masaryk Memorial Cancer Institute, Brno, Czech Republic

e Department of Pediatric Oncology, University Hospital Brno and Department of Laboratory Medicine, Masaryk Memorial Cancer Institute, Zluty kopec 7, Brno, Czech Republic
E-mail: valik@mou.cz
Fax: +420 543 136 721
Tel: +420 543 136 700

Abstract

To improve recovery, selectivity and reproducibility of SELDI-TOF analyses, we found it necessary to modify manufacturer's recommended protocols on sample and chip preparation. To yield reproducible denaturing conditions we verified concentrations of denaturing, reducing and lipid-solubilizing agents. We improved sorption of molecules of interest and reproducibility of analyses by introducing the preconditioning step and alkaline/acidic elutions for normal phase chips. The ratio that reproducibly decomposed the specimen was urea 9 mol l−1 + DTT 10 mmol l−1 + CHAPS 20 g l−1. For sample denaturation, 100 µl of the fresh mixture was added to 100 µl of the specimen. Our modification of a chip processing increased recovery of the NP20 chip by up to 400% as assessed by total ion current. We obtained the range of mass accuracy of 0.02–0.04% and response precision between 30–40% of m/z+. We observed about 50% peak overlap. To obtain approximately 92% of possible peaks three chip selectivities, IMAC, H50 and normal phase with alkaline wash should be used. The selectivity of the SELDI chips is affected by unspecific interactions of a sample with a chip backbone. The system is compatible with matrix-based biological materials and does not suffer from urea interference and sensitivity to covalently bound alkaline ions. The technique is reasonably suitable for semiquantitative screening in the mammalian low-molecular weight cellular, tissue and plasma proteome.

Graphical abstract: When one chip is not enough: Augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens

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Article information

DOI
https://doi.org/10.1039/B815503H
Article type
Technical Note
Submitted
05 Sep 2008
Accepted
12 Dec 2008
First published
15 Jan 2009

Lab Chip, 2009,9, 1014-1017

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When one chip is not enough: Augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens

G. Kristina, P. Radomir, B. Eva, D. Lenka, L. Radek, V. Rostislav, V. Borivoj and V. Dalibor, Lab Chip, 2009, 9, 1014 DOI: 10.1039/B815503H

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