2009
When one chip is not enough: Augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens
GREPLOVÁ, Kristína, Radomír PILNÝ, Eva BUDINSKÁ, Lenka DUBSKÁ, Radek LAKOMÝ et. al.Základní údaje
Originální název
When one chip is not enough: Augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens
Název česky
Když jeden čip nestačí: Zvyšování úrovně validity SELDI-TOF proteomických profilů klinických vzorků.
Autoři
GREPLOVÁ, Kristína, Radomír PILNÝ, Eva BUDINSKÁ, Lenka DUBSKÁ, Radek LAKOMÝ, Rostislav VYZULA, Bořivoj VOJTĚŠEK a Dalibor VALÍK
Vydání
Lab on a Chip, 2009, 1473-0197
Další údaje
Jazyk
angličtina
Typ výsledku
Článek v odborném periodiku
Obor
30200 3.2 Clinical medicine
Stát vydavatele
Česká republika
Utajení
není předmětem státního či obchodního tajemství
Odkazy
Impakt faktor
Impact factor: 6.306
Organizační jednotka
Lékařská fakulta
UT WoS
000264256200024
Klíčová slova anglicky
SELDI-TOF; proteomic profile; validity; chip
Štítky
Příznaky
Mezinárodní význam, Recenzováno
Změněno: 24. 4. 2009 13:36, Mgr. Eva Budinská, Ph.D.
V originále
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.
Česky
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.
Návaznosti
| LC06035, projekt VaV |
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| MZ0MOU2005, záměr |
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| NR8338, projekt VaV |
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