FIG. 3.

Relative quantification of PAP I's interacting partners using guanidination. (A) Workflow of the guanidination procedure, described in detail in Materials and Methods. After in-gel digestion with trypsin, the guanidination reaction was carried out using differentially labeled N-O-methylisourea. The wild-type peptides were labeled with ¹⁴N, which shifts the mass of lysine-containing peptides by +42 Th, and the ΔsprE peptides were labeled with ¹⁵N, for a shift of +44 Th. After the samples were mixed, relative quantification was performed by comparing the intensities of the light and heavy monoisotopic peaks. A parallel experiment was performed using reverse labeling. IP, immunopurification. (B) Sample spectra of guanidinated PAP I and HrpA at the MS and MS/MS level. On the MS panels, representative arginine-containing peptides are labeled. The T*⁺ designation represents the tryptic fragments containing modified lysine residues. The enlarged regions show the peptide of interest, with the +2-Th shift between heavy and light peaks. The insets in the MS/MS spectra illustrate the +2-Th shift between y ions. K* refers to homoarginine. (C) The relative quantification of PAP I interacting partners in the ΔsprE background. Since the +2-Th-shifted peak represents a mixture of the monoisotopic ΔsprE peptides and the third isotope of the wild-type peaks, all intensities of the heavy peptides were adjusted using the formula (ΔsprE_obs − ISO_exp) + [PAP I^WT_obs − (PAP I^ΔSprE_obs − ISO_exp)], where ΔsprE_obs is the intensity of any peptide from the ΔsprE samples (obs, observed) and ISO_exp is the predicted isotopic peak intensity (exp, expected). All intensities were adjusted to that of PAP I to correct for differences in starting material (see Fig. S3 in the supplemental material). (D) Results of the reverse labeling experiment, where wild-type peptides were derivatized with [¹⁵N]O-methylisourea and ΔsprE peptides with [¹⁴N]O-methylisourea. The isotopic correction described in Fig. S3 in the supplemental material was therefore not necessary. The intensities of the peaks from the wild-type peptides were set at 100%, and the intensities of the ΔsprE peptides are reported relative to that. All intensities were adjusted to that of PAP I to correct for differences in starting material. Error bars show standard deviations. WT, wild type.