Large-Scale Identification of Phosphorylation Sites for Profiling Protein Kinase Selectivity

Protein kinase selectivity is largely governed by direct binding to the target site(s) on the substrate. Thus, substrate determinants identified from sequences around phosphorylation sites are desirable resources for matching kinases to their substrates. In this study, we tried to identify kinase-selective substrate determinants, including motif sequences, based on large-scale discovery of kinase/substrate pairs. For this purpose, we employed a combination strategy of <i>in vitro</i> kinase reaction followed by LC–MS/MS analysis and applied it to three well-studied kinases: c-AMP regulated protein kinase A (PKA), extracellular signal-regulated kinase 1 (ERK1), and RAC-alpha serine/threonine-protein kinase (AKT1). Cellular proteins were fractionated, dephosphorylated with thermosensitive alkaline phosphatase, phosphorylated with the target kinase, and digested with Lys-C/trypsin, and then phosphopeptides were enriched using TiO₂-based hydroxy acid-modified metal oxide chromatography (HAMMOC) and subjected to LC–MS/MS. As a result, 3585, 4347, and 1778 <i>in vitro</i> phosphorylation sites were identified for PKA, ERK1, and AKT1, respectively. As expected, these extensive identifications of phosphorylation sites enabled extraction of both known and novel motif sequences, and this in turn permitted fine discrimination of the specificities of PKA and AKT1, which both belong to the AGC kinase family. Other unique features of the kinases were also characterized, including phospho-acceptor preference (Ser or Thr) and bias ratio of singly/multiply phosphorylated peptides. More motifs were found with this methodology as compared with target kinase phosphorylation of peptides obtained by predigestion of proteins with Lys-C/trypsin. Thus, this approach to characterization of kinase substrate determinants is effective for identification of kinases associated with particular phosphorylation sites

Extended Coverage of Singly and Multiply Phosphorylated Peptides from a Single Titanium Dioxide Microcolumn

We developed a novel approach to enlarge phosphoproteome coverage by selective elution depending on the number of phosphoryl group of peptides from a single titanium dioxide (TiO₂) microcolumn using hydrophilic interaction chromatography (HILIC). In this approach, acidic methylphosphonate buffer including organic solvent is used for selective elution of singly phosphorylated peptides from an aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) microcolumn and basic elution conditions with phosphate, ammonium hydroxide, and pyrrolidine are then employed for eluting multiply phosphorylated peptides retained by the HAMMOC microcolumn. Finally, we successfully identified 11 300 nonredundant phosphopeptides from triplicate analyses of 100 μg of HeLa cell lysates using this approach. This simple strategy made it possible to accomplish comprehensive and efficient phosphoproteome analysis from limited sample amounts loaded onto a single HAMMOC microcolumn without additional fractionation or enrichment approaches

Extended Coverage of Singly and Multiply Phosphorylated Peptides from a Single Titanium Dioxide Microcolumn

We developed a novel approach to enlarge phosphoproteome coverage by selective elution depending on the number of phosphoryl group of peptides from a single titanium dioxide (TiO₂) microcolumn using hydrophilic interaction chromatography (HILIC). In this approach, acidic methylphosphonate buffer including organic solvent is used for selective elution of singly phosphorylated peptides from an aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) microcolumn and basic elution conditions with phosphate, ammonium hydroxide, and pyrrolidine are then employed for eluting multiply phosphorylated peptides retained by the HAMMOC microcolumn. Finally, we successfully identified 11 300 nonredundant phosphopeptides from triplicate analyses of 100 μg of HeLa cell lysates using this approach. This simple strategy made it possible to accomplish comprehensive and efficient phosphoproteome analysis from limited sample amounts loaded onto a single HAMMOC microcolumn without additional fractionation or enrichment approaches

Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins

During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset

Acetic Acid Ion Pairing Additive for Reversed-Phase HPLC Improves Detection Sensitivity in Bottom-up Proteomics Compared to Formic Acid

Despite the general acceptance of formic acid as the additive of choice for peptide reversed-phase LC-MS/MS applications, some still argue that the selection of acetic acid represents a better option. To settle this debate, we investigated both the difference in MS sensitivity and chromatographic behavior of peptides between these two systems. This interlaboratory study was performed using different MS setups and C18 separation media employing both 0.1% formic and 0.5% acetic acid as ion pairing modifiers. Relative to formic acid, we find an overall ∼2.2–2.5× increase in MS signal and a slight decrease in RP LC retention (−0.7% acetonitrile on average) for acetic acid conditions. While these two features have opposing effects on peptide detectability, we find that acetic acid produces up to 60% higher peptide ID output depending on the type of sample. The drop in RPLC retention increases with peptide net charge at acidic pH. MS signal is dependent on the difference between the charge of the precursor ion and the charge of the peptide in solution, favoring species with a low pI. Lower peptide retention under acetic acid conditions demonstrates its higher hydrophilicity and, as expected, leads to composition and sequence-dependent character of the observed retention shift

FliC phosphorylation affects type 2 secretome levels in static biofilms.

<p>(A) Type 2 secretome analysis for static biofilms of PAO1 WT, Δ<i>fliC</i>, Δ<i>fliC</i>-FL T27A, Δ<i>fliC</i>-FL and Δ<i>fliC</i>-FL S28A strains grown in 6-well plate for 24 h. Experiment was conducted with three biological replicates and two technical replicates each. (B) Quantification of proteases from (A) by Image J 1.43 software (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) showing increase in representative T2SS proteases of Δ<i>fliC</i>—FL T27A and Δ<i>fliC</i>—FL S28A. All differences are significant with student’s t-test p-values < 0.05.</p

Influence of FliC phosphorylation on dynamic biofilms formed under flow cell conditions.

<p>Comparison of biofilm architecture in confocal-ortho view for PAO1 WT, Δ<i>fliC</i>, Δ<i>fliC</i>-FL T27A, Δ<i>fliC</i>-FL and Δ<i>fliC</i>-FL S28A strains across 7 days. Live and dead cells are represented in green and red, respectively. Panels are represented as a-WT, b-Δ<i>fliC</i>, c-Δ<i>fliC</i>- FL T27A, d-Δ<i>fliC</i>-FL and e-Δ<i>fliC</i>-FL S28A, respectively. Magnification is under 40X oil lens. Scale bars indicate a distance of 50 μm.</p

FliC phosphomutants have delayed dispersal.

<p>Total biovolumes of PAO1 WT, Δ<i>fliC</i>, Δ<i>fliC</i>-FL T27A, Δ<i>fliC</i>-FL and Δ<i>fliC</i>-FL S28A strains measured over a 7 day flow cell experiment. Reduction in biovolume from day 4 to day 5 in WT and Δ<i>fliC</i>- FL and from day 6 to day 7 in Δ<i>fliC</i>- FL T27A and Δ<i>fliC</i>- FL S28A is observed. Error bars represent mean ± SD for three biological replicates.</p

FliC phosphorylation affects T2SS secretion efficiency.

<p>(A) Immunoblot of extracellular LasB (top panel), intracellular LasB (middle panel) and intracellular RNA polymerase (RNA Pol) α-subunit (bottom panel) at 13 h for PAO1 WT, Δ<i>fliC</i>, Δ<i>fliC</i>-FL T27A, Δ<i>fliC</i>-FL and Δ<i>fliC</i>-FL S28A strains. Proteins were loaded based on equal number of cells as shown by RNA Pol α-subunit levels (bottom panel). (B) Quantification of extracellular LasB levels at 13 h in Δ<i>fliC</i>-FL T27A vs. Δ<i>fliC</i>-FL and Δ<i>fliC</i>-FL S28A vs. Δ<i>fliC</i>–FL. Error bars represent mean ±SD calculated from five biological replicates. Student’s t-test p-values < 0.05 for Δ<i>fliC</i>-FL T27A vs. Δ<i>fliC</i>–FL and Δ<i>fliC</i>-FL S28A vs. Δ<i>fliC</i>-FL. (C) Immunoblot of membrane proteins XcpY, XcpP, XcpQ in PAO1 WT, Δ<i>fliC</i>, Δ<i>fliC</i>-FL T27A, Δ<i>fliC</i>-FL and Δ<i>fliC</i>-FL S28A strains at 13 h. The proteins were loaded from equal number of bacterial cells as shown by immunoblotting of RNA Pol α-subunit levels.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p