Liquid Chromatographic Analysis and Mass Spectrometric Identification of Farnesylated Peptides

Farnesylation involves the post-translational attachment of a 15 carbon unit to the C-terminus of proteins, thus allowing them to incorporate into membranes. The farnesylation reaction requires farnesyldiphosphate as the farnesyl group donor and is catalyzed by the farnesyltransferase. Some of the most familiar farnesylated proteins belong to the Ras protein superfamily, well-known oncoproteins. As Ras proteins require the membrane localization for the transduction of extracellular signals, farnesyltransferase inhibitors are discussed as chemotherapeutic agents. Despite the importance of this post-translational modification, farnesylated peptides have been investigated rarely by means of high-pressure liquid chromatography in combination with mass spectrometry. In this study, we examined the liquid chromatographic separation of farnesylated peptides with the help of the multidimensional protein identification technology. The peptides were further ionized by electrospray ionization and subsequently analyzed by tandem mass spectrometry. We demonstrated that farnesylated peptides are more strongly retained by reversed phase than nonfarnesylated peptides. This allowed for the identification of farnesylated peptides, if spiked into complex peptide samples. In some cases the farnesyl group was apparently split off from the peptide during the ionization process, and tandem mass spectra often revealed a neutral loss of the farnesyl moiety

Multidimensional Protein Identification Technology-Selected Reaction Monitoring Improving Detection and Quantification for Protein Biomarker Studies

The targeted analysis of proteins in complex biological samples is best achieved using selected reaction monitoring (SRM). To maximize the sensitivity of this approach, sample fractionation or enrichment is still required, particularly to detect less abundant proteins in clinically relevant biofluids. Here, we report the development of multidimensional protein identification technology (MudPIT)-SRM, taking advantage of the robust online strong cation exchange chromatography for tryptic peptide fractionation and combining it with the multiplexed, quantitative attributes of SRM. The classical MudPIT method has been modified with an in-line strategy to introduce reference peptides onto the analytical column to enable quantitation at each salt step. Applying the MudPIT-SRM approach to profile abundant plasma proteins, we demonstrated mean increases in peak areas of almost 90% compared to conventional SRM. MudPIT-SRM analyses of low abundant proteins present in human wound fluid exudates similarly demonstrated increased peak areas and enabled the detection of proteins which were below the lower limit of detection when analyzed by conventional SRM. The MudPIT-SRM method is relatively facile to conduct and offers performance advantages to enhance sensitivity for biomarker studies

Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway, Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

<div><p>Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus <i>Sordaria macrospora</i>. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated <i>mik1</i> gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.</p></div

PRO40 is required for correct signaling via the CWI pathway.

<p>(A) Time course of MAK1 phosphorylation in <i>pro40</i> deletion (Δ; S69656) and overexpression strains (OE; T184.2NS11) in comparison to wildtype. Strains were grown for three to six days, and phosphorylated MAK1 was detected in a Western blot using an anti-phospho-p44/42 antibody. The signal for tubulin was used as internal standard. Representative immunoblots of two to four independent experiments with three technical replicates are shown. (B) Stress-induced MAK1 phosphorylation in <i>pro40</i> deletion (Δ; S69656) and overexpression strains (OE; T184.2NS11) in comparison to wildtype. Strains were grown for three days and subjected to 0.01% H₂O₂ for 0, 15, 30, and 45 minutes prior to harvesting. Phosphorylated MAK1 was detected using an anti-phospho-p44/42 antibody, and the signal for tubulin was used as internal standard. Representative immunoblots of three independent experiments with three technical replicates are shown. (C) Model of the scaffolding function of PRO40 for the CWI pathway. Details are discussed in the text.</p

Localization of MIK1, MEK1, and MAK1.

<p>(A) GFP-labeled MIK1 and MEK1 are present in the cytoplasm and absent from nuclei. GFP-labeled MAK1 localizes to the cytoplasm, but is also targeted to the nucleus. (B) Co-localization experiments with MEK1-GFP and H2B-tdTomato show that spherical organelles devoid of MEK1 labeling are nuclei (arrowheads). Scale bar, 10 µm.</p

Shared interaction network of MEK1 and PRO40.

<p>Venn diagram comparing the three datasets generated from affinity purification and mass spectrometry with strains Δpro40::PRO40-FLAG (PRO40), Δmek1::NTAP-MEK1 (MEK1), and Δpro40::NTAP-MEK1 (MEK1(Δpro40)). The 12 proteins found in all three datasets and the 17 proteins found only in the PRO40 and MEK1 datasets are represented by boxes with <i>S. macrospora</i> locus tag numbers or protein designations. The magenta and blue box color indicates that the transcript of the encoding gene belongs to the top500 transcripts (with respect to read counts) in protoperithecia and vegetative hyphae, respectively (data taken from a previous transcriptomics analysis <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004582#pgen.1004582-Teichert1" target="_blank">[41]</a>).</p