Translate your question into comparisons
Define the variables you intend to compare, such as treatment states or experimental systems. Groups should address a clear question; more samples alone do not make a complete design.
Use controls to assess background
Chemical labeling and affinity enrichment may generate background signals. Appropriate negative controls help assess nonspecific enrichment and interpret treatment-group changes. Choose controls for the experimental system.
Distinguish biological and technical replicates
Biological replicates assess differences and consistency among independent samples. Technical replicates assess measurement stability. They answer different questions and are not interchangeable. Agree on replication during study planning.
Plan interpretation in advance
Before collection, define whether you need identification, differential enrichment, quantitative comparisons or target validation. These goals impose different design, quality and validation requirements.