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Effects of scanning sensitivity and multiple scan algorithms on microarray data quality

ABSTRACT: A number of methods have been proposed to incorporate multiple scans at different intensities to reduce the quantification error and to minimize effects of saturation, but no direct comparison of their efficacy has been made. Here we used 40 technical replicates to compare individual scans at low, medium and high sensitivity with three methods for combining data from multiple scans (either 2 scan or 3 scan cases) using a variety of metrics Keywords: Multiple Scans

ORGANISM(S): Mus musculus

PROVIDER: GSE19493 | GEO | 2010/03/22

SECONDARY ACCESSION(S): PRJNA122287

REPOSITORIES: GEO

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Project description:Untargeted metabolomics aims at obtaining quantitative information on the highest possible number of low-molecular biomolecules present in a biological sample. Rather small changes in mass spectrometric spectrum acquisition parameters may have a significant influence on the detectabilities of metabolites in untargeted global-scale studies by means of high-performance liquid chromatography-mass spectrometry (HPLC-MS). Employing whole cell lysates of human renal proximal tubule cells, we present a systematic global-scale study of the influence of mass spectrometric scan parameters on the number and intensity of metabolites detectable in whole cell lysates. Ion transmission and ion collection efficiencies in an Orbitrap-based mass spectrometer generally depend on the m/z range scanned, which, ideally, requires different instrument settings for the respective mass ranges investigated. Therefore, we split a full scan range of m/z 50-1000 relevant for metabolites into two separate segments (m/z 50-200 and m/z 200-1000), allowing an independent tuning of the ion transmission parameters for both mass ranges. Three different implementations, involving either scanning from m/z 50-1000 in a single scan, or scanning from m/z 50-200 and from m/z 200-1000 in two alternating scans, or performing two separate HPLC-MS runs with m/z 50-200 and m/z 200-1000 scan ranges were critically assessed. The most efficient approach in terms of feature number, which forms the basis for statistical analysis, identification, and for generating biological hypotheses, was the separate analysis of two different mass ranges. This lead to an increase in the number of detectable metabolite features, especially in the higher mass range (m/z greater than 400), by 2.5 (negative mode) to 6-fold (positive mode) as compared to analysis involving a single scan range. Application of the method to metabolite extracts prepared from whole cell lysates of human renal proximal tubule epithelial cells initially yielded up to 13,000 – 69,000 detectable features, which were subjected to rigorous background filtering and quality control in order to obtain reliable metabolite features for subsequent differential quantification.

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Effects of scanning sensitivity and multiple scan algorithms on microarray data quality

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