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Statement of the problem
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• Specify comparisons of interest
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• Express comparisons as statistical hypotheses
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• Define scope of biological replication
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• Restricted scope suitable for screening; expanded scope required for validation
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Exploratory data analysis
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• Detect mis-identified features
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• Remove obvious outliers
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• Detect features with missing values
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• Choose imputation strategy
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Model-based analysis
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• Fit linear mixed model per protein
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• Reduced scope of biological replication = fixed subjects; expanded scope = random subjects
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• Check qq-plots plots for Normality
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• If deviations, conclusions are approximate only
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• Check residual plots for equal variance
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• If deviations, use iterative least squares
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• Test comparisons of interest
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• Adjust p-values per comparison to control FDR
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• Quantify protein abundance in conditions or samples of interest
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• Use as input with downstream clustering or classification
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Design follow-up experiments
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• Evaluate power and sample size
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• Find minimal sample size for a fold change
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• Find minimal fold change for a sample size
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