Christine Enjalbal

Proteomics-based Refinement of Deinococcus deserti Genome Annotation Reveals an Unwonted Use of Non-canonical Translation Initiation Codons

Deinococcaceae are a family of extremely radiation-tolerant bacteria that are currently subjected to numerous studies aimed at understanding the molecular mechanisms for such radiotolerance. To achieve a comprehensive and accurate annotation of the Deinococcus deserti genome, we performed an N terminus-oriented characterization of its proteome. For this, we used a labeling reagent, N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide, to selectively derivatize protein N termini. The large scale identification of N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide-modified N-terminal-most peptides by shotgun liquid chromatography-tandem mass spectrometry analysis led to the validation of 278 and the correction of 73 translation initiation codons in the D. deserti genome. In addition, four new genes were detected, three located on the main chromosome and one on plasmid P3. We also analyzed signal peptide cleavages on a genome-wide scale. Based on comparative proteogenomics analysis, we propose a set of 137 corrections to improve Deinococcus radiodurans and Deinococcus geothermalis gene annotations. Some of these corrections affect important genes involved in DNA repair mechanisms such as polA, ligA, and ddrB. Surprisingly, experimental evidences were obtained indicating that DnaA (the protein involved in the DNA replication initiation process) and RpsL (the S12 ribosomal conserved protein) translation is initiated in Deinococcaceae from non-canonical codons (ATC and CTG, respectively). Such use may be the basis of specific regulation mechanisms affecting replication and translation. We also report the use of non-conventional translation initiation codons for two other genes: Deide_03051 and infC. Whether such use of non-canonical translation initiation codons is much more frequent than for other previously reported bacterial phyla or restricted to Deinococcaceae remains to be investigated. Our results demonstrate that predicting translation initiation codons is still difficult for some bacteria and that proteomics-based refinement of genome annotations may be helpful in such cases.

Mass spectrometry in combinatorial chemistry

In the fast expanding field of combinatorial chemistry, profiling libraries has always been a matter of concern--as illustrated by the buoyant literature over the past seven years. Spectroscopic methods, including especially mass spectrometry and to a lesser extent IR and NMR, have been applied at different levels of combinatorial library synthesis: in the rehearsal phase to optimize the chemistry prior to library generation, to confirm library composition, and to characterize after screening each structure that exhibits positive response. Most of the efforts have been concentrated on library composition assessment. The difficulties of such analyses have evolved from the infancy of the combinatorial concept, where large mixtures were prepared, to the recent parallel syntheses of collections of discrete compounds. Whereas the complexity of the analyses has diminished, an increased degree of automation was simultaneously required to achieve efficient library component identification and quantification. In this respect, mass spectrometry has been found to be the method of choice, providing rapid, sensitive, and informative analyses, especially when coupled to chromatographic separation. Fully automated workstations able to cope with several hundreds of compounds per day have been designed. After a brief introduction to describe the combinatorial approach, library characterization will be discussed in detail, considering first the solution-based methodologies and secondly the support-bound material analyses.

Comparison of inert supports in laser desorption/ionization mass spectrometry of peptides: pencil lead, porous silica gel, DIOS-chip and NALDI target.

In the search for alternative inert surfaces replacing silicon chips in Desorption/Ionization On porous Silicon (DIOS)-like mass spectrometry analyses, nanostructured silicon-based NALDI chips were evaluated in Laser Desorption/Ionization (LDI) of peptides. Comparisons were made using commercially available DIOS chips (MassPREP-DIOS-target), amorphous carbon powder from lead pencil and porous silica gel used for chromatographic purposes as reference supports. A set of synthetic model peptides presenting variable amino acid sequences of various lengths was analyzed under all conditions. The LDI responses of the four matrix-free techniques were compared, especially in terms of peptide detection sensitivity and overall experiment robustness.

Studies of selective Boc removal in the presence of silyl ethers

Abstract The selective removal of N-Boc protection can be obtained in the presence of either TBDMS or TBDPS ethers. On the basis of promising results from the literature, we first tried sonication, that failed, whereas the exclusive cleavage of the Boc group was successfully achieved by a saturated solution of HCl in ethyl acetate.

Laser desorption ionization mass spectrometry of protein tryptic digests on nanostructured silicon plates.

We report on the simple application of a new nanostructured silicon (NanoSi) substrate as laser desorption/ionization (LDI)-promoting surface for high-throughput identification of protein tryptic digests by a rapid MS profiling and subsequent MS/MS analysis. The NanoSi substrate is easily prepared by chemical etching of crystalline silicon in NH(4)F/HNO(3)/AgNO(3) aqueous solution. To assess the LDI performances in terms of sensitivity, repeatability and robustness, the detection of small synthetic peptides (380-1700Da) was investigated. Moreover, peptide sequencing was tackled. Various tryptic synthetic peptide mixtures were first characterized in MS and MS/MS experiments carried out on a single deposit. Having illustrated the capability to achieve peptide detection and sequencing on these ionizing surfaces in the same run, protein tryptic digests from Cytochrome C, β-Casein, BSA and Fibrinogen were then analyzed in the femtomolar range (from 50 fmol for Cytochrome C down to 2 fmol for Fibrinogen). Comparison of the NanoSi MS and MS/MS data with those obtained with sample conditioned in organic matrix demonstrated a great behavior for low mass responses. We demonstrated the capability of LDI on NanoSi to be a complementary method to MALDI peptide mass fingerprinting ensuring determination of peptide molecular weights and sequences for more efficient protein database searches.

Investigation of Silicon-Based Nanostructure Morphology and Chemical Termination on Laser Desorption Ionization Mass Spectrometry Performance

We have evaluated the laser desorption ionization mass spectrometry (LDI-MS) performance of six nanostructured silicon surfaces of different morphologies and chemical functionalizations. The substrates have been synthesized either by metal-assisted etching method or by vapor-liquid-solid (VLS) growth technique. In addition to the commercial nanostructured silicon-based surface (NALDI) target plates, serving as reference, the homemade surfaces have been evaluated in mass spectrometry experiments conducted with peptide solutions mimicking tryptic digests. LDI surfaces synthesized by metal-assisted etching method were the most efficient in terms of signal intensities and number of detected peptides. The surface providing the best LDI-MS performance was composed of two nanostructured layers. Interestingly, we also observed a significant influence of the type of organic coating (hydrocarbon vs fluorocarbon) on peptide ionization discrimination.

Quantitative determination of rocuronium in human plasma by liquid chromatography–electrospray ionization mass spectrometry

Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was used for the quantification of the neuromuscular blocking agent rocuronium in human plasma. Verapamil was used as internal standard. The samples were subjected to a dichloromethane liquid-liquid extraction after ion pairing of the positively charged ammonium compound with iodide prior to LC-MS. Optimized conditions involved separation on a Symmetry Shield RP-18 column (50 x 2.1 mm, 3.5 microm) using a 15-min gradient from 10 to 90% acetonitrile in water containing 0.1% trifluoroacetic acid at 250 microl/min. Linear detector responses for standards were observed from 25 to 2,000 ng/ml. The extraction recovery averaged 59% for rocuronium and 83% for the internal standard. The limit of quantification (LOQ), using 500 microl of plasma, was 25 ng/ml. Precision ranged from 1.3 to 19% (LOQ), and accuracy was between 92 and 112%. In plasma samples, at 20 and 4 degrees C, rocuronium was stable at physiological pH for 4 h; frozen at -30 degrees C it was stable for at least 75 days. The method was found suitable for the analysis of samples collected during pharmacokinetic investigations in humans.

Laser Desorption/Ionization Mass Spectrometry on Porous Silica and Alumina for Peptide Mass Fingerprinting

We investigated a variant of desorption/ionization on porous silicon (DIOS) mass spectrometry utilizing an aqueous suspension of either porous silica gel or porous alumina (pore size of 60 and 90 Å, respectively). Laser desorption/ionization (LDI) from samples directly deposited on a stainless steel surface without any inorganic substrates was also achieved. Synthetic peptides designed to cover large sequence diversity constituted our model compounds. Sample preparation, including material conditioning, peptide solubilization, and deposition protocol onto standard matrix-assisted laser desorption/ionization (MALDI) probe, as well as ionization source tuning were optimized to perform sensitive reproducible LDI analyses. The addition of either a cationizing agent or an alkali metal scavenger to the sample suspension allowed modification of the ionization output. Comparing hydrophilic silica gel to hydrophobic reversed-phase silica gel as well as increasing material pore size provided further insights into desorption/ionization processes. Furthermore, mixtures of peptides were analyzed to probe the spectral suppression phenomenon when no interfering organic matrix was present. The results gathered from synthetic peptide cocktails indicated that LDI mass spectrometry on silica gel or alumina constitutes a promising complementary method to MALDI in proteomics for peptide mass fingerprinting.

Tof-SIMS Analysis of Polymer Bound Fmoc-protected Peptides

Abstract We report the results obtained in the step by step control of a solid phase peptide synthesis carried out on Sheppard polyamide resin by Time of flight - Secondary Ion Mass Spectrometry.

A new generation of cross-linkers for selective detection by MALDI MS

We designed a new cross‐linker bearing a CHCA moiety. The use of the CHCA‐tagged cross‐linker JMV 3378 in conjunction with a neutral MALDI matrix α‐cyano‐4‐hydroxycinnamic methyl ester enabled specific signal enhancement in MALDI‐TOF MS of cross‐link containing peptides. Discrimination between modified and non‐modified peptides can be achieved by comparison of two spectra, one using CHCA and the other using the α‐cyano‐4‐hydroxycinnamic methyl ester matrix. The methodology was validated using cytochrome c and apo‐myoglobine as model proteins.