Search

We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with [K.sub.d] < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have ~3-8-fold higher affinity and ~2-4-foId higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions. high-throughput DNA sequencing | PDGF-BB | Quantitative Selection of Aptamers through Sequencing | MicroMagnetic Separation device doi/ 10.1073/pnas.1009331107

How the primary sequence of a protein encodes conformational preferences that operate early in folding to promote efficient formation of the correct native topology is still poorly understood. To address this issue, we have prepared a set of yeast iso-1-cytochrome c variants that contain polyalanine inserts ranging from 6 to 30 residues in length near the N terminus of the protein. We study the thermodynamics and kinetics of His-heme loop formation in the denatured state at 3 and 6 M guanidine-HCl concentration. We find that polyalanine closely approximates a random coil with excluded volume giving scaling exponents, [v.sub.3], for equilibrium loop formation of 2.26 [+ or -] 0.13 and 1.97 [+ or -] 0.04 in 3 and 6 M guanidine-HCl, respectively. The rate of loop breakage initially decreases and then becomes independent of loop size as would be expected for a random coil. Comparison with previously reported data for denatured state His-heme loop formation for iso-1-cytochrome c and Rhodopseudomonas palustris cytochrome c', shows that foldable sequences deviate significantly from random coil behavior and that the deviation is fold-dependent. protein folding | residual structure | chain stiffness doi/ 10.1073/pnas.1004 572107

The primary structure of a 13.6 kDa single heavy chain camelid antibody ([V.sub.H]H) was determined by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) top-down sequence analysis. The majority of the sequence was obtained by mass spectrometric de novo sequencing, with the N-terminal 14 amino acid residues being determined using [T.sup.3]-sequencing and database interrogation. The determined sequence was confirmed by liquid chromatography--tandem mass spectrometry (LC--MS/MS) analysis of a tryptic digest, which also provided high-energy collisionally induced dissociation (CID) data permitting the clear assignment of 3 of the 14 isobaric Leu/Ile residues. Five of the 11 Leu/Ile ambiguities could be resolved by homology comparisons with known [V.sub.H]H sequences. The monoisotopic molecular weight of the [V.sub.H]H was determined by ultrahigh-resolution orthogonal electrospray (ESI)-TOF analysis and found to be 13 610.6066 Da, in excellent agreement with the established sequence. To our knowledge, this is the first time that the entire primary structure of a protein with a molecular weight > 13 kDa has been established by mass spectrometric top-down sequencing. 10.1021/ac1000515

It has previously been shown that photodissociation of tryptic peptide ions with 157 nm light in a matrix-assisted laser desorption/ionizaton (MALDI) tandem time-of-flight (TOF) mass spectrometer generates an abundance of x-type ions. A peptide de novo sequencing algorithm has now been developed to interpret these data. By combination of photodissociation and postsource decay (PSD) spectra, the algorithm identifies x-type ions and derives peptide sequences. The confidence of amino acid assignments is evaluated by observing complementary y-, v-, and w-type ions that provide additional constraints to sequence identification. In the analysis of 31 tryptic peptides from 4 model proteins, the algorithm identified 322 (or 90.7%) of the 355 amino acids and made only 3 incorrect assignments. The other 30 amino acids were not identified because specific needed x-type ions were not detected. Based on the observation of v- and w-type ions, 45 of 50 detected leucine and isoleucine residues were successfully distinguished and there was only one mistake. The remaining four residues were not distinguished because the corresponding v- and w-type ions were not detected. These de novo sequencing results translated into successful identification of proteins through homology searches. To evaluate the robustness of the present sequencing approach, a collection of 266 tryptic peptides from 23 model proteins were analyzed and then sequenced. A total of 167 peptides yielded sequence tags of 5 or more residues. In 5 peptides, 1 or 2 residues were incorrectly assigned. 10.1021/ac902050y

AAA+ proteases are ATP-fueled machines that bind protein substrates via a degradation tag, unfold the molecule if necessary, and then translocate the polypeptide into a chamber for proteolysis. Tag recognition is normally viewed as a passive reaction. By contrast, for the AAA+ Lon protease, we show that degron tags are also regulatory elements that determine protease activity levels. Indeed, different tags fused to the same protein change degradation speeds and energetic efficiencies by 10-fold or more. Degron binding to multiple sites in the Lon hexamer appears to differentially stabilize specific enzyme conformations, including one with high protease and low ATPase activity, and results in positively cooperative degradation. These allosteric mechanisms allow Lon to operate in either a fast or slow proteolysis mode, according to specific physiological needs, and may help maximize degradation of misfolded proteins following stress-induced denaturation. AAA+ protease | allosteric control | degradation tags www.pnas.org/cgi/doi/10.1073/pnas.0910392106

Problem statement: Protein sequence clustering is a method used to discover relations between proteins. This method groups the proteins based on their common features. It is a core process in protein sequence classification. Graph theory has been used in protein sequence clustering as a means of partitioning the data into groups, where each group constitutes a cluster. Mohseni-Zadeh introduced a maximal cliques algorithm for protein clustering. Approach: In this study we adapted the maximal cliques algorithm of Mohseni-Zadeh to find cliques in protein sequences and we then parallelized the algorithm to improve computation times and allowed large protein databases to be processed. We used the N-Gram Hirschberg approach proposed by Abdul Rashid to calculate the distance between protein sequences. The task farming parallel program model was used to parallelize the enhanced cliques algorithm. Results: Our parallel maximal cliques algorithm was implemented on the stealth cluster using the C programming language and a hybrid approach that includes both the Message Passing Interface (MPI) library and POSIX threads (PThread) to accelerate protein sequence clustering. Conclusion: Our results showed a good speedup over sequential algorithms for cliques in protein sequences. Key words: Maximal cliques algorithm, parallel processing, protein sequence clustering, MPI, pthread, INTRODUCTION One of the basic applications of protein sequence comparison is in protein sequence clustering. Protein sequence clustering is an element of protein sequence analysis. The results of protein sequence [...]

ATP7B is a copper-transporting P-type ATPase present predominantly in liver. In basal copper, hepatic ATP7B is in a post-trans-Golgi network (TGN) compartment where it loads cytoplasmic Cu(I) onto newly synthesized ceruloplasmin. When copper levels rise, the protein redistributes via unique vesicles to the apical periphery where it exports intracellular Cu(I) into bile. We want to understand the mechanisms regulating the copper-sensitive trafficking of ATP7B. Earlier, our laboratory reported the presence of apical targeting/TGN retention information within residues 1-63 of human ATP7B; deletion of these residues resulted in a mutant protein that was not efficiently retained in the post-TGN in low copper and constitutively trafficked to the basolateral membrane of polarized, hepatic WIF-B cells with and without copper (13). In this study, we used mutagenesis and adenovirus infection of WIF-B cells followed by confocal immunofluorescence microscopy analysis to identify the precise retention/targeting sequences in the context of full-length ATP7B. We also analyzed the expression of selected mutants in livers of copper-deficient and -loaded mice. Our combined results clearly demonstrate that nine amino acids, [F.sub.37]AFDNVGY[E.sub.45], comprise an essential apical targeting determinant for ATP7B in elevated copper and participate in the TGN retention of the protein under low-copper conditions. The signal is novel, does not require phosphorylation, and is highly conserved in -24 species of ATP7B. Furthermore, N41S, which is part of the signal we identified, is the first and only Wilson disease-causing missense mutation in residues 1--63 of ATP7B. Expression of N41S-ATP7B in WIF-B cells severely disabled the targeting and retention of the protein. We present a working model of how this physiologically relevant signal might work. Wilson protein; WIF-B cells; mutagenesis; in vivo; trans-Golgi network

Tandem mass spectrometry is the prevailing approach for large-scale peptide sequencing in high-throughput proteomic profiling studies. Effective database search engines have been developed to identify peptide sequences from MS/MS fragmentation spectra. Since proteins are polymorphic and subject to post-translational modifications (PTM), however, computational methods for detecting unanticipated variants are also needed to achieve true proteome-wide coverage. Different from existing 'unrestrictive' search tools, we present a novel algorithm, termed SIMS (for Sequential Motif Interval Search), that interprets pairs of product ion peaks, representing potential amino acid residues or 'intervals', as a means of mapping PTMs or substitutions in a blind database search mode. An effective heuristic software program was likewise developed to evaluate, rank, and filter optimal combinations of relevant intervals to identify candidate sequences, and any associated PTM or polymorphism, from large collections of MS/MS spectra. The prediction performance of SIMS was benchmarked extensively against annotated reference spectral data sets and compared favorably with, and was complementary to, current state-of-the-art methods. An exhaustive discovery screen using SIMS also revealed thousands of previously overlooked putative PTMs in a compendium of yeast protein complexes and in a proteome-wide map of adult mouse cardiomyocytes. We demonstrate that SIMS, freely accessible for academic research use, addresses gaps in current proteomic data interpretation pipelines, improving overall detection coverage, and facilitating comprehensive investigations of the fundamental multiplicity of the expressed proteome.

De novo sequencing is a spectrum analysis approach for mass spectrometry data to discover post-translational modifications in proteins; however, such an approach is still in its infancy and is still not widely applied to proteomic practices due to its limited reliability. In this work, we describe a de novo sequencing approach for the discovery of protein modifications based on identification of the proteome UStags (Shen, Y.; Tolic, N.; Hixson, K. K.; Purvine, S. O.; Pasa-Tolic, L.; Qian, W. J.; Adkins, J. N.; Moore, R. J.; Smith, R. D. Anal. Chem. 2008, 80, 1871-1882). The de novo information was obtained from Fourier-transform tandem mass spectrometry data for peptides and polypeptides from a yeast lysate, and the de novo sequences obtained were selected based on filter levels designed to provide a limited yet high quality subset of UStags. The DNA-predicted database protein sequences were then compared to the UStags, and the differences observed across or in the UStags (i.e., the UStags' prefix and suffix sequences and the UStags themselves) were used to infer possible sequence modifications. With this de novo--UStag approach, we uncovered some unexpected variances within several yeast protein sequences due to amino acid mutations and/or multiple modifications to the predicted protein sequences. To determine false discovery rates, two random (false) databases were independently used for sequence matching, and ~3% false discovery rates were estimated for the de novo--UStag approach. The factors affecting the reliability (e.g., existence of de novo sequencing noise residues and redundant sequences) and the sensitivity of the approach were investigated and described. The combined de novo--UStag approach complements the UStag method previously reported by enabling the discovery of new protein modifications.

In Bio-Informatics application, the analysis of protein sequence is a kind of computation driven science which has rapidly and quickly growing biological data. Also databases used in these applications are heterogeneous in nature and alignment of protein sequence using physical techniques is expensive, slow and results are not always guaranteed/accurate. So this application requires cross-platform, cost-effective and more computing power algorithm for sequence matching and searching a sequence in database. Grid is one of the most emerging technologies of cost effective computing paradigm for large class of data and compute intensive application which enables large-scale aggregation and sharing of computational data and other resources across institutional boundaries. We proposed the Grid architecture for searching of distributed, heterogeneous genomic databases which contained protein sequences to speed up the analysis of large scale sequence data and performed sequence alignment for residues match. Key words: Grid computing, bio-informatics, sequence alignment, dynamic programming, INTRODUCTION Grid computing, most simply stated, is distributed computing taken to the next evolutionary level. The goal is to create the illusion of a simple yet large and powerful self [...]

Leucyi-tRNA synthetase (LeuRS) has evolved an editing function to clear misactivated amino acids. An Escherichia coli-based assay was established to identify amino acids that compromise the fidelity of LeuRS and translation. Multiple nonstandard as well as standard amino acids were toxic to the cell when LeuRS editing was inactivated.

We present new statistical models of peptide fragment peak intensities that achieve unprecedented accuracy, and we discuss their application to the problem of scoring in database searching or peptide de novo sequencing. The models, made available via open-source computer programs, are very general and can potentially improve any intensity-based scoring.

We describe a new method for encoded synthesis, efficient on-resin screening, and rapid unambiguous sequencing of combinatorial peptide libraries. An improved binary tag system for encoding peptide libraries during synthesis was designed to facilitate unequivocal assignment of isobaric residues by MALDI-TOF MS analysis. The improved method for encoded library synthesis was combined with a new versatile on-resin screening strategy that permitted multiple stages and types of screening to be employed successively on one library under mild conditions. The new method facilitated a combinatorial study of transglutaminase (TGase) enzyme substrate peptides, revealing new details of the effect of amino acid composition on TGase substrates. The approach was first demonstrated for an encoded library (130 321 compounds) of lysine pentapeptide substrates of TGase, synthesized using the 'split-mix' method. The library was reacted on-resin with TGase enzyme and a soluble desthiobiotin labeled glutamine substrate. Initial screening was performed by adsorbing streptavidin-coated magnetic microparticles onto library beads, followed by magnetic separation. The differential binding affinities of desthiobiotin and biotin for streptavidin were exploited to release the magnetic microparticles and regenerate the desthiobiotin-labeled resin beads for further screening by flow-cytometry-based automated bead sorting, resulting in 345 beads that were sequenced by MALDI-TOF MS analysis. A second library consisted of encoded glutamine hexapeptide substrates, which was reacted on-resin with TGase enzyme and a soluble desthiobiotin-labeled cadaverine. Two-stage screening identified 267 glutamine peptides as TGase-reactive, of which 21 were further analyzed by solution-phase enzyme kinetics. Kinetic results indicated that the peptide PQQQYV from the library has a 68-fold greater substrate specificity than the best known glutamine substrate QQIV. The new encoding and screening strategies described here are expected to be broadly applicable to synthesis and screening of combinatorial peptide libraries in the future.

To identify a simplified code for conformational switching, we have redesigned two natural proteins to have 88% sequence identity but different tertiary structures: a 3-[alpha] helix fold and an [alpha]/[beta] fold. We describe the design of these homologous heteromorphic proteins, their structural properties as determined by NMR, their conformational stabilities, and their affinities for their respective ligands: IgG and serum albumin. Each of these proteins is completely folded at 25[degrees]C, is monomeric, and retains the native binding activity. The complete binding epitope for both ligands is encoded within each of the proteins. The IgG-binding epitope is functional only in the [alpha]/[beta] fold, and the albumin-binding epitope is functional only in the 3-[alpha] fold. These results demonstrate that two monomeric folds and two different functions can be encoded with only 12% of the amino acids in a protein (7 of 56). The fact that 49 aa in these proteins are compatible with both folds shows that the essential information determining a fold can be highly concentrated in a few amino acids and that a very limited subset of interactions in the protein can tip the balance from one monomer fold to another. This delicate balance helps explain why protein structure prediction is so challenging. Furthermore, because a few mutations can result in both new conformation and new function, the evolution of new folds driven by natural selection for alternative functions may be much more probable than previously recognized. evolution | folding | NMR | protein design | protein structure

Tandem mass spectrometry (MS/MS) has become the experimental method of choice for high-throughput proteomics-based biological discovery. The two primary ways of analyzing MS/MS data are database search and de novo sequencing. In this paper, we present a new approach to peptide de novo sequencing, called MSNovo, which has the following advanced features. (1) It works on data generated from both LCQ and LTQ mass spectrometers and interprets singly, doubly, and triply charged ions. (2) It integrates a new probabilistic scoring function with a mass array-based dynamic programming algorithm. The simplicity of the scoring function, with only 6-10 parameters to be trained, avoids the problem of overfitting and allows MSNovo to be adopted for other machines and data sets easily. The mass array data structure explicitly encodes all possible peptides and allows the dynamic programming algorithm to find the best peptide. (3) Compared to existing programs, MSNovo predicts peptides as well as sequence tags with a higher accuracy, which is important for those applications that search protein databases using the de novo sequencing results. More specifically, we show that MSNovo outperforms other programs on various ESI ion trap data. We also show that for high-resolution data the performance of MSNovo improves significantly. Supporting Information, executable files and data sets can be found at http:// msms.usc.edu/supplementary/msnovo.

An improved method for peptide de novo sequencing by MALDI mass spectrometry is presented. The method couples a charge derivatization reaction with C-terminal digestion to modify tryptic peptides. The charge derivatization attaches a fixed charge group onto the N-termini of peptides, and the enzymatic digestion after the derivatization step removes C-terminal basic amino acid residues such as arginine and lysine. The fragmentation of the modified peptide(s) under low-energy CID conditions (MALDI Q-TOF mass spectrometer) yields a simplified yet complete ion series of the peptide sequence. The validity of the method is demonstrated by the results from several model protein digests, where peptide sequences were correctly deduced either manually or through an automated sequencing program.

Here, we describe a method for protein identification and de novo peptide sequencing. Through in vivo cell culturing, the deuterium-labeled lysine residue (Lys-[d.sub.4]) introduces a 4-Da mass tag at the carboxyl terminus of proteolytic peptides when cleaved by certain proteases. The 4-Da mass difference between the unlabeled and the deuterated lysine assigns a mass signature to all lysine-containing peptides in any pool of proteolytic peptides for protein identification directly through peptide mass mapping. Furthermore, it was used to distinguish between N-and C-terminal fragments for accurate assignments of daughter ions in tandem MS/MS spectra for sequence assignment. This technique simplifies the labeling scheme and the interpretation of the MS/MS spectra by assigning different series of fragment ions correctly and easily and is very useful in de novo peptide sequencing. We have also successfully implemented this approach to the analysis of protein mixtures derived from the human proteome.

By using sequence information from an aligned protein family, a procedure is exhibited for finding sites that may be functionally or structurally critical to the protein. Features based on sequence conservation within subfamilies in the alignment and associations between sites are used to select the sites. The sites are subject to statistical evaluation correcting for phylogenetic bias in the collection of sequences. This method is applied to two families: the phycobiliproteins, light-harvesting proteins in cyanobacteria, red algae, and cryptomonads, and the globins that function in oxygen storage and transport. The sites identified by the procedure are located in key structural positions and merit further experimental study.

Research was conducted to determine the partial structure of the yeast La protein and compare it with human protein. Structural analysis indicates that the domain of 113-amino acids, involved in the RNA-binding, differs from human La.

Automated protein sequencers are still being used in protein laboratories despite competition from mass spectrometers. One such sequencer, HP 241 (N +C) is produced by Hewlett-Packard Bioscience Products. It may be automated or manually controlled and employs high performance liquid chromatography. Three sequencers, namely Pocise HT, Procise cLC and Procise C, are made by Perkin-Elmer Applied Biosystems Division. They all utilize a Macintosh PowerMac G3 control system and a Sequence Pro Data System for Windows/Macintosh.

A new technique for determining sequence and linkage information of underivatized oligosaccharides is developed using alkaline degradation and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS). Alkaline degradation (also known as the 'peeling' reaction) is a chemical degradation technique that only cleaves the glycosidic bond at the reducing end by [Beta]-elimination to yield a new reducing end. The reaction products are sampled directly with minimal cleanup and monitored by MALDI-FTMS to elucidate the oligosaccharide sequence. Linkage information is provided by cross-ring cleavage fragmentation of the new reducing ends, created by either MALDI source fragmentation or sustained off-resonance irradiation collision-induced dissociation. This method is illustrated by the successful sequence and linkage determination of neutral, branched, fucosylated, and sialylated oligosaccharides. Experiments on differently linked disaccharides are also performed to determine the specificity of the cross-ring cleavage reactions. The power of this technique is enhanced by the Fourier transform mass analyzer, which provides high-resolution, exact mass, and facile tandem mass spectrometry experiments of MALDI-produced ions.

Very small quantifies of glycoproteins were directly processed on a MALDI sampling plate prior to their mass spectrometric investigations. The on-plate digestion with N-glycanase released effectively the corresponding oligosaccharides in very short times, irrespective of their molecular mass. The following treatment with an array of exoglycosidase enzymes enables sequencing and a linkage-form determination in analysis times that are considerably shorter than achieved previously: the entire structural determination on a glycoprotein can be completed in one day, with a minimum substrate consumption. Ribonuclease B, bovine fetuin, human [[Alpha].sub.1]-acid glycoprotein, and the diamine oxidase (from porcine kidney) have been used to illustrate different aspects of the on-plate sample treatment/MALDI mass spectrometry.

The enantiomeric separation of phenylthiocarbamoyl derivatives of amino acids (PTC-AAs) was studied on a series of reversed phase HPLC columns coupled to the chiral phase HPLC columns. First, the five chiral phases (native, 0.2, 3.3, 7.5 and 16.9 phenylcarbamoylated/[Beta]-cyclodextrins, Ph/CD) were newly prepared by modification of [Beta]-cyclodextrin with phenyl isocyanate and were examined for the enantiomeric separation of PTC-AAs. Among them, the 3.3Ph/CD phase gave the best enantiomeric separation ([Alpha] [greater than or equal to] 1.04). However, the separation of the individual PTC-AAs was not sufficient. Next, these separations were investigated on various reversed phase HPLC columns, and octyl silica was selected in terms of the suitability of the mobile phase adopted for the enantiomeric separation mentioned above. The effects of the column temperature, the ion-pairing reagent, and the final content of methanol were also studied on the tandem column of octyl silica and the 3.3Ph/CD phase. Under the best conditions (100 mM ammonium acetate (pH 6.5) containing 1 mM butanesulfonate with 0-40% methanol as the mobile phase), all the individual PTC-AAs were well separated within 150 min. The applicability of the method was demonstrated by the sequence/configuration analysis of a peptide containing a o-amino acid ([D-[Thr.sup.2]]-leucine enkephalin-Thr).

Matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometry is shown to be a powerful tool for the elucidation of protein modifications. Low-energy covalent bonds that originate from certain posttranslational modifications dissociate preferentially to produce characteristic mass spectrometric signatures that prove useful for the accurate, confident identification and characterization of such modifications. Because the MALDI ion trap is an authentic tandem mass spectrometer, it proves feasible to acquire secondary information to test hypotheses as to the nature and site of the putative modifications - further increasing the reliability of the tool. The method combines the advantageous features of MALDI (i.e., the ability to measure the same sample repeatedly, to measure unfractionated complex mixtures without the need for sample cleaning, and to determine peptide mixtures with subpicomole sensitivity) with the ease and the speed of the ion trap measurement. We demonstrate how the unique properties of MALDI ion trap MS can be used to address problems involving the determination of both native posttranslational modifications of proteins (e.g., disulfide mapping, glycosylation determination, and phosphorylation determination) and non-native chemical modifications of proteins (e.g., methionine oxidation and photo-cross-linking of proteins with DNA).

A procedure is described for rapid, high-confidence identification of proteins using matrix-assisted laser desorption/ionization tandem ion trap mass spectrometry in conjunction with a genome database searching strategy. The procedure involves excision of copper-stained bands or spots from electrophoretic gels, in-gel trypsin digestion of the proteins, single-stage mass spectrometric analysis of the resultant mixture of tryptic peptides, followed by tandem ion trap mass spectrometric analysis of selected individual peptides, and database searching of the relevant genomic database using the program PepFrag. The scheme provides sensitive, real-time protein identification as well as facile identification of modifications. A single operator can unambiguously identify 5-10 proteins/day from an organism whose genome is known at a level of >0.5 pmol of protein loaded on a gel. The utility of the technique was demonstrated by the identification and characterization of a band from a human HTLV-I preparation and 11 different proteins from a yeast RNA polymerase II C-terminal repeat domain-affinity preparation. The technology has great potential for postgenome biological science, where it promises to facilitate the dissection and anatomy of macromolecular assemblages, the definition of disease state markers, and the investigation of protein targets in biological processes such as the cell cycle and signal transduction.

Sequencing conditions for postsource decay and collision-induced dissociation/postsource decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have been optimized to elucidate the structure of polyglycylation of tubulin. This posttranslational modification involves the linkage of multiple glycine residues through the [Gamma]-carboxyl of glutamic acid residues in the carboxyl termini of the protein. Individual [Alpha]- and [Beta]-tubulin polypeptides contain respectively three and four potential glycylation sites. The sample preparation we used was the thin-layer preparation of the target specimen in the presence of [Alpha]-cyano-4-hydroxycinnamic acid and nitrocellulose. The study of different synthetic polyglycylated peptides fragmentation (modified peptides with the linear sequence 427DATAEEEGEFEEEGE[Q.sup.441]) shows that the peptides fragment regularly to form major fragments of b- and y-type ions with negligible side-chain fragmentation. The rules were applied to the structural elucidation of a Paramecium [Beta]-tubulin hexaglycylated peptide available in the subpicomole range. Polyglycylation was identified on the last four glutamic acid residues.

A new technique for sequencing proteins isolated by polyacrylamide gel electrophoresis is proposed. The method uses nano-electrospray tandem mass spectrometry. The technique is around 10-100 times more sensitive, faster and requires less purification and manipulation steps than other existing techniques. It is expected to become the method of choice for protein microcharacterization.

Phosphorylation of guanosine and inosine is catalysed by the same enzyme thus facilitating the construction of delta gsk strains which can help in cloning mutant E. coli gsk alleles and heterologous guanosine kinase encoding genes. The study clones the gsk gene of E. coli and concludes that this gene is structural gene for guanosine kinase. Guanosine and inosine utilization of the gsk-1 mutant strain is restored by the cloned gene.

The hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) isdiscussed. Anthrobacter sp. strains and TM1 (12) were used in this physiological and chemical study wherein dehalogenase genes were subjected to genetic analysis. These genes were cloned and expressed in Escherechia coli. The nucleotide sequence of the 4-CBA region was also determined and presented. Various degrees of homology were found between genes, proteins and amino acids.

Cf-plasma desorption mass spectrometry (PDMS) was used to study the fragmentation of ribonuclease A, papain and proteinase K, all of which are within the 13- to 29-kDa mass range. The results showed that the plasma desorption process could induce efficient, sequence-specific fragmentation of large, native proteins. The procedure could be used as a starting step in the sequence determination of large protein molecules.

Although much is known about microRNAs' regulation in gene expression and their contributions in cell fate, to date, globally lineage-(cell-) specific identification of the binding events between a transcription factor and its targeting microRNA genes is still waiting for elucidation. In this paper, we performed a ChIP-Seq experiment to find the targeting microRNA genes of a transcription factor, Egr1, in human erythroleukemia cell line K562. We found Egr1 binding sites near the promoters of 124 distinct microRNA genes, accounting for about 42% of the miRNAs which have high-confidence predicted promoters (294). We also found EGR1 bind to another 63 pre-miRNAs. We chose 12 of the 187 microRNAs with Egr1 binding sites to perform ChlP-PCR assays and the positive binding signal from ChlP-PCR confirmed the ChIP-Seq results. Our experiments provide the first global binding profile between Egr1 and its targeting microRNA genes in PMA-treated K562 cells, which may facilitate the understanding of pathways controlling microRNA biology in this specific cell line., 1. Introduction MicroRNAs (miRNAs) are a family of ~22-nucleotide small noncoding RNAs in eukaryotes and mainly involve in regulation at posttranscriptional level by translational repression or degradation their target mRNAs [...]