Yet-Ran Chen

Immobilized metal affinity chromatography revisited: pH/acid control toward high selectivity in phosphoproteomics.

Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, a highly specific enrichment protocol is still a challenge in large-scale studies. Here, we report a simple pH/acid control method that addresses the poor specificity seriously criticized in IMAC. Detailed evaluation of the capture and release mechanism in IMAC revealed that pH, buffer and salt yield a complex interplay in enrichment of phosphopeptides, yet they play individual roles in recovery and specificity. A revised one-step IMAC method with low sample loss and high specificity can be rationally designed by controlling salt, pH and the structure and concentration of organic acid. Without methyl esterification, the one-step IMAC enrichment with single LC-MS/MS identified 386 phosphoproteins in 550 mug of non-small-cell lung cancer cell lysate with 96% specificity. Additional fractionation by SDS-PAGE from 4 mg of cell lysate revealed the comprehensive proteome map, identifying 2747 phosphorylation sites from 2360 nondegenerate phosphopeptides and 1219 phosphoproteins with a false discovery rate of 0.63%. To our knowledge, this pH/acid-controlled IMAC procedure provides higher specificity than any other one-step IMAC purification procedure. Furthermore, the simple and reproducible IMAC protocol can be adapted to other solid supports, fully automated or manual, for large-scale identification of the vastly under-explored phosphoproteome.

Identification of Downstream Components of Ubiquitin-Conjugating Enzyme PHOSPHATE2 by Quantitative Membrane Proteomics in Arabidopsis Roots

This work reports quantitative membrane proteomic analysis and provides insights into the mechanism of PHO2-mediated Pi acquisition via posttranslational regulation of PHT1s. The results highlight the fundamental role of PHO2 in orchestrating Pi acquisition at the root surface and Pi loading at the xylem of root stele to maintain Pi homeostasis. MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/PHOSPHATE2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ (for isobaric tags for relative and absolute quantitation)- based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in PHOSPHATE TRANSPORTER1 (PHT1) family and PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research.

Analysis of coptisine, berberine and palmatine in adulterated Chinese medicine by capillary electrophoresis-electrospray ion trap mass spectrometry.

Chinese medicine preparations contaminated with coptisine, berberine and palmatine were studied by capillary electrophoresis-electrospray ion trap mass spectrometry. The dubious adulterants were identified by their retention times, molecular ions and specific fragment ions produced from collision induced dissociation. The results showed that, in comparison with CE-UV and capillary electrophoresis-electrospray mass spectrometry (CE-ESI-MS), more reliable identification could be achieved with CE-ESI-MS-MS using ion trap mass spectrometry.

A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis

Highlight An 11 aa peptide derived from one of the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 (CAP) superfamily is salt regulated, conferring salt susceptibility through suppression of salt-tolerance genes.

Deficiency of NPGPx, an oxidative stress sensor, leads to obesity in mice and human

Elevated oxidative stress is closely associated with obesity. Emerging evidence shows that instead of being a consequence of obesity, oxidative stress may also contribute to fat formation. Nonselenocysteine‐containing phospholipid hydroperoxide glutathione peroxidase (NPGPx) is a conserved oxidative stress sensor/transducer and deficiency of NPGPx causes accumulation of reactive oxygen species (ROS). In this communication, we show that NPGPx was highly expressed in preadipocytes of adipose tissue. Deficiency of NPGPx promoted preadipocytes to differentiate to adipocytes via ROS‐dependent dimerization of protein kinase A regulatory subunits and activation of CCAAT/enhancer‐binding protein beta (C/EBPβ). This enhanced adipogenesis was alleviated by antioxidant N‐acetylcysteine (NAC). Consistently, NPGPx‐deficient mice exhibited markedly increased fat mass and adipocyte hypertrophy, while treatment with NAC ablated these phenotypes. Furthermore, single nucleotide polymorphisms (SNPs) in human NPGPx gene, which correlated with lower NPGPx expression level in adipose tissue, were associated with higher body mass index (BMI) in several independent human populations. These results indicate that NPGPx protects against fat accumulation in mice and human via modulating ROS, and highlight the importance of targeting redox homeostasis in obesity management.

Quantitative Peptidomics Study Reveals That a Wound-Induced Peptide from PR-1 Regulates Immune Signaling in Tomato

CAPE1, a conserved peptide elicitor derived from tomato PR-1, was induced by wounding and found to regulate immune responses against biological threats. As PR-1 is highly conserved across many organisms and the putative peptide from AtPR1 was also found to be bioactive in Arabidopsis, the results suggest that this peptide may be useful for enhancing resistance to stress in other plant species. Many important cell-to-cell communication events in multicellular organisms are mediated by peptides, but only a few peptides have been identified in plants. In an attempt to address the difficulties in identifying plant signaling peptides, we developed a novel peptidomics approach and used this approach to discover defense signaling peptides in plants. In addition to the canonical peptide systemin, several novel peptides were confidently identified in tomato (Solanum lycopersicum) and quantified to be induced by both wounding and methyl jasmonate (MeJA). A wounding or wounding plus MeJA-induced peptide derived from the pathogenesis-related protein 1 (PR-1) family was found to induce significant antipathogen and minor antiherbivore responses in tomato. This study highlights a role for PR-1 in immune signaling and suggests the potential application of plant endogenous peptides in efforts to defeat biological threats in crop production. As PR-1 is highly conserved across many organisms and the putative peptide from At-PR1 was also found to be bioactive in Arabidopsis thaliana, our results suggest that this peptide may be useful for enhancing resistance to stress in other plant species.

Analysis of triazines by reversed electroosmotic flow capillary electrophoresis–electrospray mass spectrometry

Abstract Normal and reversed electroosmotic flow (EOF) capillary electrophoresis (CE) has been studied for the analysis of eight triazine herbicides. Reversed EOF CE, dynamically modified with cetyltrimethylammonium bromide (CTAB), provided better separation than normal CE. All eight triazines were baseline resolved with the use of 50 mM ammonium acetate and 0.7 mM CTAB as the running buffer. The method of sheath liquid was used in the on-line coupling of CE with electrospray mass spectrometry. The composition of sheath liquid was found to be critical in the coupling of reversed EOF CE with MS. The resolution obtained in CE–UV can be preserved in CE–MS if the composition of the sheath liquid is similar to the running buffer.

Tunnel frit: a nonmetallic in-capillary frit for nanoflow ultra high-performance liquid chromatography-mass spectrometryapplications.

In this study, an easy method to fabricate a durable in-capillary frit was developed for use in nanoflow liquid chromatography (nanoLC). A small orifice was tunneled into the sol-gel frit during the polymerization process resulting in the simple fabrication of a tunnel frit. A short packing tunnel frit column (2 cm, C(18) particles) was able to sustain over 10,000 psi continuous liquid flow for 10 days without observation of particle loss, and back pressure variation was less than 5%. The tunnel frit was successfully applied to the fabrication of nanoflow ultra high-performance liquid chromatography (nano-UHPLC) trap and analytical columns. In the analysis of tryptic peptides, the tunnel frit trap and analytical columns were demonstrated to have high separation efficiency and sensitivity. In analysis of phosphopeptides, the use of the nonmetallic tunnel frit column showed better sensitivity than the metallic frit column. This design can facilitate the preparation of nano-HPLC and nano-UHPLC columns and the packing material can easily be refilled when the column is severely contaminated or clogged.

Metabolite identification for mass spectrometry-based metabolomics using multiple types of correlated ion information.

Metabolite identification remains a bottleneck in mass spectrometry (MS)-based metabolomics. Currently, this process relies heavily on tandem mass spectrometry (MS/MS) spectra generated separately for peaks of interest identified from previous MS runs. Such a delayed and labor-intensive procedure creates a barrier to automation. Further, information embedded in MS data has not been used to its full extent for metabolite identification. Multimers, adducts, multiply charged ions, and fragments of given metabolites occupy a substantial proportion (40-80%) of the peaks of a quantitation result. However, extensive information on these derivatives, especially fragments, may facilitate metabolite identification. We propose a procedure with automation capability to group and annotate peaks associated with the same metabolite in the quantitation results of opposite modes and to integrate this information for metabolite identification. In addition to the conventional mass and isotope ratio matches, we would match annotated fragments with low-energy MS/MS spectra in public databases. For identification of metabolites without accessible MS/MS spectra, we have developed characteristic fragment and common substructure matches. The accuracy and effectiveness of the procedure were evaluated using one public and two in-house liquid chromatography-mass spectrometry (LC-MS) data sets. The procedure accurately identified 89% of 28 standard metabolites with derivative ions in the data sets. With respect to effectiveness, the procedure confidently identified the correct chemical formula of at least 42% of metabolites with derivative ions via MS/MS spectrum, characteristic fragment, and common substructure matches. The confidence level was determined according to the fulfilled identification criteria of various matches and relative retention time.

Pregnenolone activates CLIP-170 to promote microtubule growth and cell migration

Pregnenolone (P5) is a neurosteroid that improves memory and neurological recovery. It is also required for zebrafish embryonic development. However, its mode of action is unclear. Here we show that P5 promotes cell migration and microtubule polymerization by binding a microtubule plus end-tracking protein, cytoplasmic linker protein 1 (CLIP-170). We captured CLIP-170 from zebrafish embryonic extract using a P5 photoaffinity probe conjugated to diaminobenzophenone. P5 interacted with CLIP-170 at its coiled-coil domain and changed it into an extended conformation. This increased CLIP-170 interaction with microtubules, dynactin subunit p150(Glued) and LIS1; it also promoted CLIP-170-dependent microtubule polymerization. CLIP-170 was essential for P5 to promote microtubule abundance and zebrafish epiboly cell migration during embryogenesis, and overexpression of the P5-binding region of CLIP-170 delayed this migration. P5 also sustained migration directionality of cultured mammalian cells. Our results show that P5 activates CLIP-170 to promote microtubule polymerization and cell migration.