Fig. 1.

Experimental design to estimate the accuracy of label-free quantification with or without sample fractionation. The same endothelial cell lysate was loaded on a one-dimensional SDS gel and either collected in a single band or fractionated into 12 gel bands cut along the migration lane, and to assess how repeatability is affected by each step of the analytical process, we compared either nanoLC-MS/MS injection replicates or gel replicates. Four experiments were performed. A, the protein sample (15 μg) was collected in a single band and digested, and the corresponding peptide digest was analyzed three times by nanoLC-MS/MS. B, three identical protein samples (15 μg each) were loaded on the gel and collected in three bands, and after digestion, one-third of each corresponding peptide digest was analyzed once by nanoLC-MS/MS. C, the protein sample (100 μg) was fractionated by electrophoresis into 12 gel fractions, the 12 bands were digested, and each of the corresponding peptide digests was consecutively analyzed three times by nanoLC-MS/MS. D, three identical protein samples (150 μg each) were loaded on the gel and fractionated into 12 gel bands, and after digestion, the corresponding molecular weight bands of each gel lane were consecutively analyzed once by nanoLC-MS/MS (one-third of resulting peptide digests for each band).