When one chip is not enough: augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens.
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.
Kristina
Greplova
G
Department of Laboratory Medicine, Masaryk Memorial Cancer Institute, Brno, Czech Republic.
Radomir
Pilny
P
Eva
Budinska
B
Lenka
Dubska
D
Radek
Lakomy
L
Rostislav
Vyzula
V
Borivoj
Vojtesek
V
Dalibor
Valik
V
D006801:Humans; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D020543:Proteome