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@article{829396, author = {Greplová, Kristína and Pilný, Radomír and Budinská, Eva and Dubská, Lenka and Lakomý, Radek and Vyzula, Rostislav and Vojtěšek, Bořivoj and Valík, Dalibor}, article_number = {9}, keywords = {SELDI-TOF; proteomic profile; validity; chip}, language = {eng}, issn = {1473-0197}, journal = {Lab on a Chip}, title = {When one chip is not enough: Augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens}, url = {http://www.rsc.org/ej/LC/2009/b815503h.pdf}, volume = {2009}, year = {2009} }
TY - JOUR ID - 829396 AU - Greplová, Kristína - Pilný, Radomír - Budinská, Eva - Dubská, Lenka - Lakomý, Radek - Vyzula, Rostislav - Vojtěšek, Bořivoj - Valík, Dalibor PY - 2009 TI - When one chip is not enough: Augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens JF - Lab on a Chip VL - 2009 IS - 9 SP - 1014 - 1017 EP - 1014 - 1017 SN - 14730197 KW - SELDI-TOF KW - proteomic profile KW - validity KW - chip UR - http://www.rsc.org/ej/LC/2009/b815503h.pdf N2 - 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. ER -
GREPLOVÁ, Kristína, Radomír PILNÝ, Eva BUDINSKÁ, Lenka DUBSKÁ, Radek LAKOMÝ, Rostislav VYZULA, Bořivoj VOJTĚŠEK and Dalibor VALÍK. When one chip is not enough: Augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens. \textit{Lab on a Chip}. 2009, vol.~2009, No~9, p.~1014 - 1017. ISSN~1473-0197.
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