When one chip is not enough: Augmenting the validity of SELDI-TOF proteomic profiles of clinical specimens

Abstract

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 µl of the fresh mixture was added to 100 µl 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.