Distributions of phosphopeptides and their properties across 12 SCX fractions. (A) The number of phosphopeptides identified from duplicate analyses of each fraction. While similar numbers of peptides were identified in each replicate, an average of 41.3 ± 6.8% more peptides could be attributed solely to analyzing each fraction twice. (B) Venn diagram depicting the extent of overlap for the phosphopeptides identified in duplicate analyses of fraction 3. Numbers in parentheses indicate the percentage of either replicate that lies outside the overlap region. (C) Nonredundant phosphopeptides in each fraction (and replicate) with calculated solution charge states between −1 and +4. SCX separates phosphopeptides based primarily on solution charge. (D) Nonredundant phosphopeptides in each fraction containing 1 (1P) to 6 (6P) phosphorylation sites. (a and b correspond to duplicate LC–MS/MS runs of each SCX fraction).

Logo-like representations of acidic phosphorylation motifs identified by the Motif-X algorithm.21 (A) Examples of motifs extracted utilizing all 20 amino acids. Note that several permutations of a similar motif are identified. (B) Degenerate motif for casein-kinase II-like phosphorylation. Note that all three motifs in panel A are represented in this single motif. The residue at position +4 is also now significant. (C) Acidic, double phosphorylation motif identified using both degenerate analysis and considering multiple phosphorylation events. Phosphorylated serine and threonine (denoted as B and X, respectively) function as an acidic residue at position +1 in this motif.

Overlay example of phosphorylation sites from this study on a developmental pathway. The Salvador–Warts–Hippo (SWH) pathway controls organ size by modulating cell growth, proliferation, and apoptosis. Many of the genetic and biochemical interactions are known, but post-translational modifications such as phosphorylation affecting core components are almost entirely lacking. Phosphorylation events detected in this study are overlaid onto a representation of the pathway.24 Yorkie (Yki) is hyperphosphorylated in Drosophila embryos. The arrows in the figure represent what is known about the pathway from the literature where a given protein acts to stimulate or inhibit the function of the pathway. They do not indicate the direct kinase–substrate relationship.

Schematic illustration of the strategy for large-scale phosphorylation site identification from Drosophila embryos. (A) The 0–24 h old D. melanogaster w¹¹⁸ embryos were lysed and directly digested with trypsin. Tryptic peptides were desalted and then separated by SCX chromatography. Phosphopeptides from 12 SCX fractions were further enriched by IMAC and then analyzed by LC–MS/MS techniques. (B) MS/MS spectra from 24 analyses (duplicates for each sample) were searched against a composite target-decoy Drosophila protein database.11 Mass deviation, XCorr, dCn′, and solution charge state were used to filter correct from incorrect matches, maintaining <1% false-discovery rate (FDR). In total, 36 203 phosphopeptides (16 822 unique phosphopeptides) and 13 720 nonredundant phosphorylation sites were identified at a FDR of 0.63% (229 decoy matches). High-certainty localization (Ascore ≥ 13; P ≤0.05)12 was found for 10 038 sites. Finally, phosphorylation motifs (standard, degenerate, and multiply phosphorylated) were extracted from the data set with the Motif-X algorithm.21

(A) Ascore distribution for all identified sites from 36 203 peptides. Most sites could be localized with near (P ≤ 0.01) or high (P ≤ 0.05) certainty. (B) Classification of phosphorylation events into 4 general sequence categories based on kinase specificities.

Examples of motifs (represented by logo-like representations) identified in this study. The phosphorylated serine, threonine, or tyrosine are centered. (A) PKA-like substrate motifs showing a preference for arginine over lysine in degenerate analyses. (B) Novel threonine single phosphorylation motif. (C) Double phosphorylation motif (B and X represent phosphoserine and threonine, respectively) suggestive of ordered phosphorylation. (D) Examples of tyrosine single phosphorylation motifs.