Protein extract from a single mouse hippocampus was enzymatically digested and fractionated by isoelectric focusing. Aliquots of fractions were pooled to make fewer, more complex samples. The unfractionated lysate, fractions, and pooled fractions were subjected to liquid chromatography–mass spectrometry analysis. Samples consisting of many individual fractions had more protein identifications and quantified proteins with more spectral counts and greater precision than protein extract that was unfractionated or pooled into fewer LC-MS/MS samples.