Integrated Strategy for Unbiased Profiling of the Histidine Phosphoproteome

Histidine phosphorylation (pHis), which plays a key role in signal transduction in bacteria and lower eukaryotes, has been shown to be involved in tumorigenesis. Due to its chemical instability, substoichiometric properties, and lack of specific enrichment reagents, there is a lack of approaches for specific and unbiased enrichment of pHis-proteins/peptides. In this study, an integrated strategy was established and evaluated as an unbiased tool for exploring the histidine phosphoproteome. First, taking advantage of the lower charge states of pHis-peptides versus the non-modified naked peptides at weak acid solution (∼pH 2.7), strong cation exchange (SCX) chromatography was used to differentiate modified and non-modified naked peptides. Furthermore, selective enrichment of the pHis-peptide was performed by applying Cu-IDA beads enrichment. Finally, stable isotope dimethyl labeling was introduced to guarantee high-confidence assignment of pHis-peptides. Using this integrated strategy, 563 different pHis-peptides (H = 1) in 385 proteins were identified from HeLa lysates. Motif analysis revealed that pHis prefers hydrophobic amino acids and has the consensus motif-HxxK, which covered the reports from different approaches. Thus, our method may provide an unbiased and effective tool to reveal histidine phosphoproteome and to study the biological process and function of histidine phosphorylation

Integrated Strategy for Unbiased Profiling of the Histidine Phosphoproteome

Histidine phosphorylation (pHis), which plays a key role in signal transduction in bacteria and lower eukaryotes, has been shown to be involved in tumorigenesis. Due to its chemical instability, substoichiometric properties, and lack of specific enrichment reagents, there is a lack of approaches for specific and unbiased enrichment of pHis-proteins/peptides. In this study, an integrated strategy was established and evaluated as an unbiased tool for exploring the histidine phosphoproteome. First, taking advantage of the lower charge states of pHis-peptides versus the non-modified naked peptides at weak acid solution (∼pH 2.7), strong cation exchange (SCX) chromatography was used to differentiate modified and non-modified naked peptides. Furthermore, selective enrichment of the pHis-peptide was performed by applying Cu-IDA beads enrichment. Finally, stable isotope dimethyl labeling was introduced to guarantee high-confidence assignment of pHis-peptides. Using this integrated strategy, 563 different pHis-peptides (H = 1) in 385 proteins were identified from HeLa lysates. Motif analysis revealed that pHis prefers hydrophobic amino acids and has the consensus motif-HxxK, which covered the reports from different approaches. Thus, our method may provide an unbiased and effective tool to reveal histidine phosphoproteome and to study the biological process and function of histidine phosphorylation

Integrated Strategy for Unbiased Profiling of the Histidine Phosphoproteome

Histidine phosphorylation (pHis), which plays a key role in signal transduction in bacteria and lower eukaryotes, has been shown to be involved in tumorigenesis. Due to its chemical instability, substoichiometric properties, and lack of specific enrichment reagents, there is a lack of approaches for specific and unbiased enrichment of pHis-proteins/peptides. In this study, an integrated strategy was established and evaluated as an unbiased tool for exploring the histidine phosphoproteome. First, taking advantage of the lower charge states of pHis-peptides versus the non-modified naked peptides at weak acid solution (∼pH 2.7), strong cation exchange (SCX) chromatography was used to differentiate modified and non-modified naked peptides. Furthermore, selective enrichment of the pHis-peptide was performed by applying Cu-IDA beads enrichment. Finally, stable isotope dimethyl labeling was introduced to guarantee high-confidence assignment of pHis-peptides. Using this integrated strategy, 563 different pHis-peptides (H = 1) in 385 proteins were identified from HeLa lysates. Motif analysis revealed that pHis prefers hydrophobic amino acids and has the consensus motif-HxxK, which covered the reports from different approaches. Thus, our method may provide an unbiased and effective tool to reveal histidine phosphoproteome and to study the biological process and function of histidine phosphorylation

Primers for PCR amplification of genes <i>mmsB</i>, <i>tsf, and PSEEN0851</i>.

a<p>The underlined sequences are the restriction enzymes sites.</p

2-DE images of protein extracts of <i>P. putida</i> JUCT1 grown under different solvent conditions.

<p>a: nutrient medium without solvent; b: with 60% (v/v) cyclohexane; c: magnification of c1–c3, the protein spots selected in this study are circled. Arrowheads indicate the protein spots exhibiting intensity discrepancy of over 50% in samples a and b.</p

The oxidation of cyclohexane by <i>E. coli</i> strains.

<p>Incubation condition: 37°C for 8 h with cyclohexane (1%, w/v).</p

Colony formation of <i>E. coli</i> JM109 strains over-expressing <i>mmsB</i>, <i>tsf</i>, and <i>PSEEN0851</i> on LBGMg agar overlaid with decalin after 24 h incubation at 37°C. JM109 carrying empty pQE-80L was used as the control.

<p>Colony formation of <i>E. coli</i> JM109 strains over-expressing <i>mmsB</i>, <i>tsf</i>, and <i>PSEEN0851</i> on LBGMg agar overlaid with decalin after 24 h incubation at 37°C. JM109 carrying empty pQE-80L was used as the control.</p

MALDI-TOF/TOF analysis of peptides from 5 high-abundance proteins.

a<p>Number of peptides identified for individual protein in MALDI-TOF/TOF analysis.</p>b<p>Number of unique peptides identified for individual protein.</p>c<p>Total amino acid sequence of peptides identified for individual protein.</p>d<p>Percentage of amino acid sequence covered by peptides of individual protein.</p

Cell growth of recombinant <i>E. coli</i> JM109 strains in the presence of 4% (v/v) cyclohexane.

<p>The recombinant strains were cultured at 37°C and cyclohexane (CH) was added when OD₆₆₀ reached 0.2 (indicated by the arrow). □, pQE-80L (as the control); ▾, pQE-<i>mmsB</i>; •, pQE- <i>tsf</i>; △, pQE-<i>PSEEN0851</i>.</p

SDS-PAGE analysis of expression of <i>mmsB</i>, <i>PSEEN0851</i>, and <i>tsf</i> in <i>E. coli</i> JM109.

<p>The transformants were grown in LB at 37°C and induced by 1 mM IPTG. Lanes: M, molecular mass marker; 1, pQE-80L (as control); 2, pQE-<i>mmsB</i>; 3, pQE-<i>PSEEN0851</i>; 4, pQE-<i>tsf</i>. Arrowheads indicate the locations of recombinant proteins.</p