title: Considerations for powering a clinical proteomics study: Normal variability in the human plasma proteome.
abstract: Proteomics is increasingly being applied to the human plasma proteome to identify biomarkers of disease for use in non-invasive assays. 2-D DIGE, simultaneously analysing thousands of protein spots quantitatively and maintaining protein isoform information, is one technique adopted. Sufficient numbers of samples must be analysed to achieve statistical power; however, few reported studies have analysed inherent variability in the plasma proteome by 2-D DIGE to allow power calculations. This study analysed plasma from 60 healthy volunteers by 2-D DIGE. Two samples were taken, 7 days apart, allowing estimation of sensitivity of detection of differences in spot intensity between two groups using either a longitudinal (paired) or non-paired design. Parameters for differences were: two-fold normalised volume change, α of 0.05 and power of 0.8. Using groups of 20 samples, alterations in 1742 spots could be detected with longitudinal sampling, and in 1206 between non-paired groups. Interbatch gel variability was small relative to the detection parameters, indicating robustness and reproducibility of 2-D DIGE for analysing large sample sets. In summary, 20 samples can allow detection of a large number of proteomic alterations by 2-D DIGE in human plasma, the sensitivity of detecting differences was greatly improved by longitudinal sampling and the technology was robust across batches.
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, UK. d.h.jackson@leeds.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Jackson
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, UK.
authors.forename: Athula
authors.initials: A
authors.lastname: Herath
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, UK.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Swinton
authors.affiliation: Nonlinear Dynamics, Newcastle, UK.
authors.forename: David
authors.initials: D
authors.lastname: Bramwell
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, UK.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Chopra
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hughes
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, UK.
authors.forename: Kevin
authors.initials: K
authors.lastname: Cheeseman
authors.affiliation: AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, UK.|Current address: Clinical and Biomedical Proteomics Group, Cancer Research UK Clinical Centre, St. James's University Hospital, Leeds,UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Tonge
keywords: 2‐D DIGE; Image analysis; Plasma; Statistical power; Variability