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, Rostislav VYZULA, Bořivoj VOJTĚŠEK a Dalibor VALÍK. When one chip is not enough: Augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens. Lab on a Chip. 2009, roč. 2009, č. 9, s. 1014 - 1017. ISSN 1473-0197.

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

• Originální 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
• WWW: URL
• Impakt faktor: Impact factor: 6.306
• Organizační jednotka: Lékařská fakulta
• UT WoS: 000264256200024
• Klíčová slova anglicky: SELDI-TOF; proteomic profile; validity; chip
• Štítky: chip, proteomic profile, SELDI-TOF, validity
• Příznaky: Mezinárodní význam, Recenzováno

Anotace

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.

Anotace 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: Název: Centrum biofyzikální chemie, bioelektrochemie a bioanalýzy. Nové nástroje pro genomiku, proteomiku a biomedicínu.

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Centrum biofyzikální chemie, bioelektrochemie a bioanalýzy. Nové nástroje pro genomiku, proteomiku a biomedicínu

• MZ0MOU2005, záměr: Název: Funkční diagnostika zhoubných nádorů
• NR8338, projekt VaV: Název: Funkční analýza drah regulovaných proteinem p53 u nádorů prsu: Integrace genomických a proteomických přístupů zahrnujících techniky DNA-macroarray a hmotnostní spektrometrii na principu SELDI-TOF