Design and implementation of the FLEXIQuant peptide (FLEX-peptide). (a) The wheat germ transcription/translation vector highlighting the FLEX-tag. A T14K substitution (underlined) was made to generate the N-terminal tryptic FLEX-peptide (blue) for absolute quantitation experiments. The gene of interest is cloned immediately after the arginine residue. (b) The peak profile for the light (red) and heavy (blue) FLEX-peptides. The light peak intensity corresponds to 100 fmoles of spiked peptide.

Relative peptide abundances of CDC27 and APC5 in response to nocodazole exposure. The normalized light-to-heavy peak ratios (Y-axis) for all observed peptides were plotted for each time point after nocodazole exposure (0/synthesis phase, 4, 8 and 10 hrs nocodazole exposure). The peptides are drawn to scale and mapped (from the N- to C-terminus) according to their position along the protein (X-axis). (a) CDC27 - The gradual decrease of the unmodified forms of peptides spanning amino acids 197 to 446 is observed; their relative abundances drop to 10–20 % as compared to the majority of unmodified peptides, by 10 hrs of nocodazole exposure. The differences of these peptide abundances are statistically significant (asterisks). For statistical analysis, see Supplementary Figure 3. (b) APC5 - The relative abundances of three peptides (amino acids 169 to 210, asterisks) decrease dramatically (as low as 10 %) with respect to the majority of peptides. CDC27 data are averaged between two biological repeats, and a third, technical repeat (Supplementary Figure 3). APC5 data represent a single biological experiment (three technical repeats) which was done in parallel with that of one of the biological runs of CDC27. Error bars - standard deviations from the normalized ratios. For any given time point, 1 to 4 peptides were observed/quantified only two times (lacking error bars, colored in lighter shades), but were included since they were measured three times in the other time points.

The FLEXIQuant workflow comprises two stages. Stage 1 (Steps 1 to 5): Absolute quantification of the in vitro synthesized full-length labeled. The gene of interest is cloned into the WG expression vector, which is then separately transcribed and then translated in WGE (Steps 1 and 2). A defined volume of WGE expressing the protein standard is prepared for MS analysis. A known amount of (light) AQUA peptide (FLEX-peptide^L) is added prior to MS in order to quantify the protein standard via the FLEX-peptide^H (Steps 3 and 4). The concentration of the protein standard in the WGE can then be calculated based on the relative peak ratios (Step 5). Stage 2 (Steps 6 to 9) - WGE and (HeLa) cell lysate are mixed; the heavy and light proteins are co-purified and trypsinized together, resulting in identical sample preparation for both protein species (Steps 6 to 8). The absolute quantity of the light protein and a relative peptide abundance analysis can then be determined by peak integration (Step 9).

Conservation of relative light and heavy peak intensities despite artificial modifications and other stochastic events during sample preparation. A red dashed line links each monoisotopic peak to its corresponding modified form. (a) From CDC27, the elution profile/chromatogram (upper panel) and corresponding MS1 spectra (lower panel) of the unmodified peptide (i) and its deamidated forms (ii, iii). Chromatogram: the black and blue traces correspond to light and heavy profiles, respectively, and are not to scale with respect to each other. The relative monoisotopic peak ratios are indicated above each elution peak (b) From APC5, an oxidized methionine and its characteristic mass shift of 16 Da is observed for both light and heavy peptides. (c) From CDC27, the random conversion of a pyroglutamic acid from an N-terminal glutamine occurs to the same extent for both peptides. (d) From CDC27, relative peak ratios are not perturbed by incomplete trypsinization. Red stars indicate sites with decreased signal intensities. Examples are from different FLEXIQuant experiments.