When one chip is not enough: augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens
| Autor: | Vyzula Rostislav, Lakomy Radek, Dubska Lenka, Valik Dalibor, Greplova Kristina, Pilny Radomir, Vojtesek Borivoj, Budinska Eva |
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| Rok vydání: | 2009 |
| Předmět: |
Proteomics
Reproducibility Spectrometry Mass Electrospray Ionization Chromatography Proteome Chemistry Biomedical Engineering Analytical chemistry Reproducibility of Results Bioengineering Ion current Sorption General Chemistry Biochemistry Sensitivity and Specificity Matrix (chemical analysis) chemistry.chemical_compound Yield (chemistry) Urea Humans Denaturation (biochemistry) Selectivity |
| Zdroj: | Lab on a chip. 9(7) |
| ISSN: | 1473-0197 |
| Popis: | 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 microl of the fresh mixture was added to 100 microl 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. |
| Databáze: | OpenAIRE |
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