Preanalytical Impact of Sample Handling on Proteome Profiling Experiments with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Abstract

Mass spectrometry (MS) offers a wide range of possibilities for analyzing biological samples containing complex mixtures of proteins and peptides, by generating proteome profiles. The aim of most profiling experiments is identification of changes in protein patterns that are related to a certain disease or clinical status and might therefore be used to improve diagnosis, staging, and monitoring (1 ). Reproducibility of spectra generated by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS is crucial (2 ), however, and early enthusiastic reports about the diagnostic power of proteome-profiling approaches for early disease classification have recently been critically assessed and reevaluated (3 ). The reproducibility of profiling experiments depends on sample quality, which in turn is greatly influenced by preanalytical storage conditions and the choice of anticoagulants (4 -6 ). In this study, we examined the impact of preanalytical sample handling and storage times on MALDI-TOF MS data for serum and plasma samples and identified a candidate marker to allow objective classification of given samples with respect to age and, hence, appropriateness for MS protein profiling and interpretation of data.We collected 10-mL venous blood samples into serum tubes, EDTA-coated tubes, and ammonium-heparincoated tubes (Sarstedt). After collection, all samples were initially kept at room temperature for 30 min, to allow clot formation, before centrifugation at 3000g for 10 min in a precooled (4°C) centrifuge (Rotina 48R; Hettich). After centrifugation, 1 aliquot of each sample was immediately frozen at Ϫ80°C (time 0); other aliquots were incubated for distinct time periods at room temperature as follows: Specimens from 1 healthy volunteer were stored at room temperature until 1, 2, and 4 h after collection and then stored at Ϫ80°C before further use. For classification analysis, serum samples from 6 healthy volunteers and from 20 patients consecutively entering the emergency unit of the university hospital at Mannheim were collected and processed as described above, with the exception that aliquots were prepared only at time 0 and from samples stored at room temperature for 4 h after collection. The study was conducted in accordance with the regulations of the local ethics committee. All samples were deidentified.Serum and plasma samples were processed with the following magnetic bead (MB)-based affinity chromatography resins, used according to optimized protocols recommended by the manufacturer (Bruker Daltonics): hydrophobic interaction chromatography C 8 (MB-HIC-C8), weak cation-exchange chromatography (MB-WCX), and immobilized metal-ion affinity chromatography (MB-IMAC-Cu). Specifically, 5 L of the sample was mixed with the appropriate MB suspension, washed 3 times with 100 L of wash buffer, and eluted with 5 L of appropriate elution solution for the MB-HIC-C8 and MB-WCX reagent sets and 10 L of appropriate elution solution for the MB-IMAC-Cu reagent set. A portion of the eluted sample was diluted 1:...

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