We hypothesised that proteins represented in polysome fractions by nascent chains and/or co-translational complex formation would show greater intensity from N-terminal peptides in F1–F5 than in totals, as they are synthesised in the N-to-C direction. (A) Peptide midpoint calculation and example cumulative peptide intensity distribution. Steps in the cumulative intensity profiles indicate peptide midpoints. For a protein detected in polysome fractions (Fraction) through only nascent chains, a shift in signal accumulation compared with a cytoplasmic extract (Total) would be expected, such that a much greater proportion of the total peptide intensity would be expected from the N-terminal end of the protein. (B) Median number of peptides and median sequence coverage (percentage of the amino acid residues that were detected by mass spectrometry (MS) in at least one peptide) for individual proteins in unstressed samples (371 uniquely detected proteins). Proteins in the shaded area were used for the analysis (142 proteins). Cumulative peptide intensity distributions for fractions and totals of named proteins. Differences between fractions and totals were tested using the Kolmogorov–Smirnov test. (C) The proteins with significantly different fraction distributions compared with totals (p < 0.05). The fractions that are significantly different are indicated by number. Only 6 of 142 showed some evidence of a bias towards the N-terminus in one or more fractions (Asc1, Cdc60, Gpm1, Gpp1, Map1, and eIF4A/Tif2), while 5 others (Fba1, Rpl16a, Rps7a, Scp160, and eEF1A/Tef2) showed different patterns. (D) Representative examples of proteins without significant differences between fractions and totals. Dashed lines are y = x.