Proteomic analysis of low quantities of cellular material in the range obtainable from scarce patient samples

Abstract

e application of proteomics to patient material is increasingly widespread, however, a major shortcoming still are the number of cells or protein material that can be obtained. is study explores the lower limit of cell numbers that can be successfully analysed by liquid chromatography mass spectrometry to determine the protein expression pro le that is speci c to, and indicative of, the investigated cell type. e aim was to analyse an equivalent quantity of cellular material that can be obtained from, e.g., a ne-needle aspiration biopsy (FNAB). Fi een thousand and 30,000 cells from adherent (HEK293) and suspension (U937) cell lines were lysed under two di erent conditions: a 'native' and a denaturing bu er. To extend the study to clinical material, human whole PBMCs were also lysed under identical conditions. Proteins from 5,000 and 10,000 cells were analysed by both 1D and 2D-LC-MSMS on an LTQ Orbitrap XL mass spectrometer. In total, 3,219; 1,693 and 659 unique proteins were identi ed from HEK293, U937 and total PBMCs, respectively. Additionally, an iTRAQ 4-plex experiment was performed to determine the relative quantity of the proteins in the three cell types. In this study, we show that it is feasible to obtain a deep, yet cellspeci c protein pro le from a very low number of cultured and primary cells. is advancement will enable proteomic-pro ling of cellular material from ne needle aspiration biopsies that ultimately can assist cytopathologists in the diagnosis of disease.