Detailed Information on Publication Record
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.Basic information
Original name
When one chip is not enough: Augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens
Name in Czech
Když jeden čip nestačí: Zvyšování úrovně validity SELDI-TOF proteomických profilů klinických vzorků.
Authors
GREPLOVÁ, Kristína, Radomír PILNÝ, Eva BUDINSKÁ, Lenka DUBSKÁ, Radek LAKOMÝ, Rostislav VYZULA, Bořivoj VOJTĚŠEK and Dalibor VALÍK
Edition
Lab on a Chip, 2009, 1473-0197
Other information
Language
English
Type of outcome
Článek v odborném periodiku
Field of Study
30200 3.2 Clinical medicine
Country of publisher
Czech Republic
Confidentiality degree
není předmětem státního či obchodního tajemství
References:
Impact factor
Impact factor: 6.306
Organization unit
Faculty of Medicine
UT WoS
000264256200024
Keywords in English
SELDI-TOF; proteomic profile; validity; chip
Tags
Tags
International impact, Reviewed
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.
In Czech
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.
Links
| LC06035, research and development project |
| ||
| MZ0MOU2005, plan (intention) |
| ||
| NR8338, research and development project |
|