The current status of de novo sequencing of peptides by MS/MS is reviewed with focus on collision cell MS/MS spectra. The relation between peptide structure and observed fragment ion series is discussed and the exhaustive extraction of sequence information from CID spectra of protonated peptide ions is described. The partial redundancy of the extracted sequence information and a high mass accuracy are recognized as key parameters for dependable de novo sequencing by MS. In addition, the benefits of special techniques enhancing the generation of long uninterrupted fragment ion series for de novo peptide sequencing are highlighted. Among these are terminal 18 O labeling, MS n of sodiated peptide ions, N-terminal derivatization, the use of special proteases, and time-delayed fragmentation. The emerging electron transfer dissociation technique and the recent progress of MALDI techniques for intact protein sequencing are covered. Finally, the integration of bioinformatic tools into peptide de novo sequencing is demonstrated.

SummaryCentriole duplication occurs once per cell cycle and requires Plk4, a member of the Polo-like kinase family. A key component of the centrosome is the c-tubulin ring complex (c-TuRC) that nucleates microtubules. GCP6 is a member of the c-TuRC, but its role in human cells and the regulation of its functions remain unclear. Here we report that depletion of human GCP6 prevents assembly of the c-TuRC and induces a high percentage of monopolar spindles. These spindles are characterized by a loss of centrosomal c-tubulin and reduced centriole numbers. We found that GCP6 is localized in the pericentriolar material but also at distal portions of centrioles. In addition, GCP6 is required for centriole duplication and Plk4-induced centriole overduplication. GCP6 interacts with and is phosphorylated by Plk4. Moreover, we find that Plk4-dependent phosphorylation of GCP6 regulates centriole duplication. These data suggest that GCP6 is a target of Plk4 in centriole biogenesis.

Incomplete recovery from the LC column is identified as a major cause for poor detection efficiency of phosphopeptides by LC-MS/MS. It is proposed that metal ions adsorbed on the stationary phase interact with the phosphate group of phosphopeptides via an ion-pairing mechanism related to IMAC (IMAC: immobilized metal ion affinity chromatography). This may result in their partial or even complete retention. Addition of phosphate, EDTA or citrate to the phosphopeptide sample was tested to overcome the detrimental phosphopeptide suppression during gradient LC-MS/MS analysis, while the standard solvent composition (water, acetonitrile, formic acid) of the LC system was left unchanged. With the use of UPLC, a citrate additive was found to be highly effective in increasing the phosphopeptide detection sensitivity. Addition of EDTA was found to be comparable with respect to sensitivity enhancement, but led to fast clogging and destruction of the spray needle and analytical columns due to precipitation. In contrast, a citrate additive is compatible with prolonged and stable routine operation. A 50 mM citrate additive was tested successfully for UPLC-MS analysis of a commercial four-component phosphopeptide mixture, a tryptic beta-casein digest, and several digests of the 140 kDa protein SETDB1. In this protein, 27 phosphorylation sites could be identified by UPLC-MS/MS using addition of citrate, including the detection of several phosphopeptides carrying 3-5 pSer/pThr residues, compared to identification of only 10 sites without citrate addition. A 50 mM citrate additive particularly increases the recovery of multiply phosphorylated peptides, thus, extending the scope of phosphopeptide analysis by LC-MS/MS.

Characterization of all members of a gene family established by gene divergence is essential to delineate distinct or overlapping expression profiles and functionalities. Their activity as potent modulators of diverse physiological processes directs interest to galectins (endogenous lectins with b-sandwich fold binding b-galactosides and peptide motifs), warranting their study with the long-term aim of a comprehensive analysis. The comparatively low level of complexity of the galectin network in chicken with five members explains the choice of this organism as model. Previously, the three proto-type chicken galectins CG-1A, CG-1B, and CG-2 as well as the tandem-repeat-type CG-8 had been analyzed. Our study fills the remaining gap to determine gene structure, protein characteristics and expression profile of the fifth protein, that is, chimera-type chicken galectin-3 (CG-3). Its gene has a unique potential to generate variants: mRNA production stems from two promoters, alternative splicing of the form from the second transcription start point (tsp) can generate three mRNAs. The protein with functional phosphorylation sites in the N-terminus generated by transcription from the first tsp (tsp1CG-3) is the predominant CG-3 type present in adult tissues. Binding assays with neoglycoproteins and cultured cells disclose marked similarity to properties of human galectin-3. The expression and localization profiles as well as proximal promoter regions have characteristic features distinct from the other four CGs. This information on CG-3 completes the description

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It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase μLC-ICP-MS or nanoLC-ESI-MS. Using μLC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of >10 pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga(3+)-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.

The catalytic subunit of recombinant wild-type cyclic adenosine monophosphate-dependent protein kinase A (PKA) has been analyzed by a combination of 1D gel electrophoresis, in-gel digestion by trypsin, chymotrypsin, or endoproteinase AspN, and nano-ultraperformance liquid chromatography--MS/MS. The MS/MS spectra were annotated by MASCOT and the annotations were manually controlled. Using Ga(III)-immobilized metal ion affinity chromatography (IMAC), in addition to the four established autophosphorylation sites of the catalytic subunit of recombinant PKA, pSer10, pSer139, pThr197, and pSer338, six new phosphorylated residues have been characterized--pSer14, pThr48, pSer53, pSer212, pSer259, and pSer325. The established phosphorylation sites all are part of a PKA consensus motif and were found to be almost completely modified. In contrast, the newly detected sites were only partially phosphorylated. For estimation of their degree of phosphorylation, a method based on signal intensity measurements was used. For this purpose, signal intensities of all phospho- and non-phosphopeptides containing a particular site were added for estimation of site-specific phosphorylation degrees. This addition was performed over all peptides observed in the different digestion experiments, including their different charge states. pThr48 and pSer259 are located within PKA consensus motifs and were observed to be phosphorylated at 20% and 24%, respectively. pSer14 and pSer53 are located within inverted PKA consensus motifs and were found to be phosphorylated around 10% and 15%, respectively. The sequence environments of pSer212 and pSer325 have no similarity to the PKA consensus motif at all and were observed to be phosphorylated at about 5% or lower. All newly observed phosphorylation sites are located at the surface of the native protein structure of the PKA catalytic subunit. The results add new information on the theme of site-specific (auto)phosphorylation by PKA.

This study compared the effects of the beta-carboline anxiolytic, abecarnil, with other benzodiazepine receptor (BZR) ligands, including the full agonists diazepam and alprazolam, and the partial agonists ZK 95962 and bretazenil (Ro 16-6028), and alpidem, in the mouse four-plate test and plus-maze. The efficacy and potency of each compound was related to the fraction of BZR occupied by the drug. Abecarnil was efficacious in both tests and showed anxiolytic effects comparable with alprazolam and diazepam. In the four-plate test, abecarnil, bretazenil, and ZK 95962 had selective effects on releasing exploratory locomotor activity suppressed by footshock (punished crossings). None of these compounds significantly altered non-punished crossings. In contrast, diazepam and alprazolam increased both unpunished and punished crossings at low to medium doses (receptor occupancies of approximately 20-60%). The number of punished and unpunished crossings fell to control levels or below at higher, more sedative doses (approximately 80% receptor occupancy). Alpidem had very weak anxiolytic-like effects in this test and markedly reduced unpunished crossings at relatively low receptor occupancies (> 15%). In the plus-maze, abecarnil increased the time spent in the open arms and the percentage open arm entries to an extent equal to that observed following diazepam or alprazolam administration. Bretazenil and ZK 95962 had weak effects on the measures of anxiolytic activity in this test. Alpidem also had little anxiolytic-like activity in the plus-maze but markedly reduced the total number of arm entries.(ABSTRACT TRUNCATED AT 250 WORDS)