An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments

Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2–3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved.     

A procedure for amino acid sequencing in internal regions of proteins

We describe a novel procedure for determining the amino acid (aa) sequence of the internal regions of proteins. This procedure has been implemented by directly determining the sequence of aa 65-75 of the product of the trpR gene of Escherichia coli, the trp repressor. This method is based on the insertion of the cleavage site of a specific protease (factor Xa) into the protein immediately before the region to be sequenced by Edman degradation. The simplicity of the procedure makes it appealing for studies of protein structure-function relationships, and of the expression of genetic information. The method is particularly useful when there is ambiguity concerning the co-linearity of the aa and nucleotide sequences.     

TMB-Hunt: An amino acid composition based method to screen proteomes for beta-barrel transmembrane proteins

Background  

Beta-barrel transmembrane (bbtm) proteins are a functionally important and diverse group of proteins expressed in the outer membranes of bacteria (both gram negative and acid fast gram positive), mitochondria and chloroplasts. Despite recent publications describing reasonable levels of accuracy for discriminating between bbtm proteins and other proteins, screening of entire genomes remains troublesome as these molecules only constitute a small fraction of the sequences screened. Therefore, novel methods are still required capable of detecting new families of bbtm protein in diverse genomes.     

A high-yield method for the isolation of hydrophobic proteins and peptides from polyacrylamide gels for protein sequencing

A methodological approach is described which allows the isolation of hydrophobic and hydrophilic proteins and peptides in high yield. The technique consists of (1) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) protein elution from polyacrylamide gels with an organic solvent mixture composed of formic acid/acetonitrile/isopropanol/H2O (50/25/15/10, v/v/v/v), and (3) purification of eluted proteins by size exclusion chromatography on a Superose 12 column using this organic solvent mixture as eluant. The efficiency of this technique was tested with radioactively labeled polypeptides. These proteins were reaction center from Chloroflexus aurantiacus, bacteriorhodopsin, halorhodopsin from Halobacterium halobium, bovine serum albumin, ovalbumin, alpha-chymotrypsinogen A, and cytochrome c. The elution recoveries from polyacrylamide gels were 77-95%; the final yield after chromatographic purification was still 67-76% (with one exception). Subsequent amino acid sequencing was possible without further sample treatment. The sensitivity of the method described was found to be at least 20-30 micrograms protein.     

An enhanced method for peptides sequencing by N-terminal derivatization and MS

An improved method for peptide sequencing based on acetylation/deuteroacetylation in conjunction with ESI MS is introduced. Derivatization with a 1:1 mixture of acetic anhydride and deuterated acetic anhydride incorporates a stable isotope label into the analyzed molecule. This approach has been initially applied to FAB. Using MS/MS, the technique provides a fast, highly sensitive and reliable determination of the primary structure of unknown peptides. This procedure labels N-terminal fragments formed during MS/MS analysis, resulting in a simplification and faster interpretation of the spectra. The performance of the method has been tested with several synthetic peptides and applied to an efficient sequencing of the peptide map, using a nano-scale LC coupled on-line to a tandem mass spectrometer.     

C-terminal sequencing method for peptides and proteins by the reaction with a vapor of perfluoric acid in acetic anhydride

A successive C-terminal amino acid truncation reaction of peptides and proteins with a vapor generated from a low-concentrated perfluoric acid in acetic anhydride is presented. The reaction products were analyzed with matrix-assisted laser desorption/ionization-time of flight mass-spectrometry giving molecular mass ions of the C-terminal truncated peptides or proteins from which the C-terminal sequence information can be deduced. Acetylation reaction preceded the truncation reaction in order to protect the amino groups and other reactive groups in peptides and proteins, and after the truncation reaction, hydration reaction was carried out to afford cleaner mass spectra.     

An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing

Highly parallel sequencing technologies permit cost-effective whole genome sequencing of hundreds of Plasmodium parasites. The ability to sequence clinical Plasmodium samples, extracted directly from patient blood without a culture step, presents a unique opportunity to sample the diversity of "natural" parasite populations in high resolution clinical and epidemiological studies. A major challenge to sequencing clinical Plasmodium samples is the abundance of human DNA, which may substantially reduce the yield of Plasmodium sequence. We tested a range of human white blood cell (WBC) depletion methods on P. falciparum-infected patient samples in search of a method displaying an optimal balance of WBC-removal efficacy, cost, simplicity, and applicability to low resource settings. In the first of a two-part study, combinations of three different WBC depletion methods were tested on 43 patient blood samples in Mali. A two-step combination of Lymphoprep plus Plasmodipur best fitted our requirements, although moderate variability was observed in human DNA quantity. This approach was further assessed in a larger sample of 76 patients from Burkina Faso. WBC-removal efficacy remained high (<30% human DNA in >70% samples) and lower variation was observed in human DNA quantities. In order to assess the Plasmodium sequence yield at different human DNA proportions, 59 samples with up to 60% human DNA contamination were sequenced on the Illumina Genome Analyzer platform. An average ~40-fold coverage of the genome was observed per lane for samples with ≤ 30% human DNA. Even in low resource settings, using a simple two-step combination of Lymphoprep plus Plasmodipur, over 70% of clinical sample preparations should exhibit sufficiently low human DNA quantities to enable ~40-fold sequence coverage of the P. falciparum genome using a single lane on the Illumina Genome Analyzer platform. This approach should greatly facilitate large-scale clinical and epidemiologic studies of P. falciparum.     

Comparative analysis of amino acid distributions in integral membrane proteins from 107 genomes

We have performed a comparative analysis of amino acid distributions in predicted integral membrane proteins from a total of 107 genomes. A procedure for identification of membrane spanning helices was optimized on a homology-reduced data set of 170 multi-spanning membrane proteins with experimentally determined topologies. The optimized method was then used for extraction of highly reliable partial topologies from all predicted membrane proteins in each genome, and the average biases in amino acid distributions between loops on opposite sides of the membrane were calculated. The results strongly support the notion that a biased distribution of Lys and Arg residues between cytoplasmic and extra-cytoplasmic segments (the positive-inside rule) is present in most if not all organisms.     

II. Photoaffinity probes provide a general method to prepare peptide-conjugates from native protein fragments

The advantages of using amino-directed photoprobes to couple native fragments, obtained by enzymatic digestion with trypsin, to protein carriers to prepare peptide-conjugates is described. The following photoprobe reagents were investigated:N-hydroxysuccinimidylp-azidobenzoate,N-hydroxysuccinimidyl ester ofp-azidobenzoylglycine,N-hydroxysuccinimidylp-benzoylbenzoate, and pentachlorophenyl ester ofp-benzoylbenzoyl glycine or the symmetric anhydride ofp-benzoylbenzoylglycine. These reagents modify only the NH₂-terminal amino group and/or COOH-terminal -amino group of lysine of the tryptic fragments. Since the photoprobe is inert until photolysis, the probe-modified native fragment can be readily purified by high-performance liquid chromatography before cross-linking to the carrier molecule. The benzophenone photoprobes were shown to give the highest incorporation of peptide onto the protein carrier.     

Computational methods for predicting structure of membrane proteins using amino acid sequences

The structure of membrane proteins specifies their functional properties, which are important for medicine and pharmacology and, therefore, is of significant interest. The repetition of transmembrane regions that consist of hydrophobic amino acids is a characteristic and organic feature of polytopic membrane proteins. The ordered repetition (periodicity) can be detected by the Fourier method applied to a digital image of the symbolic amino acid sequence of a protein. In the present work, this investigation was carried out for 24 transmembrane proteins (successfully for 14 of them). If the repetition of transmembrane regions is aperiodic, it can be revealed by another method, that is, the method of the reiterated (four to five times) averaging of the protein hydrophobicity function in a window within the limits of 9–11 amino acids that moves along the sequence. This novel method was applied to the 24 transmembrane proteins (successfully for 19 of them) and demonstrated higher suitability than the Fourier method for predicting the secondary structure of these proteins and the corresponding functional properties.     

Important amino acid properties for enhanced thermostability from mesophilic to thermophilic proteins

Understanding the role of various interactions in enhancing the thermostability of proteins is important not only for clarifying the mechanism of protein stability but also for designing stable proteins. In this work, we have analyzed the thermostability of 16 different families by comparing mesophilic and thermophilic proteins with 48 various physicochemical, energetic and conformational properties. We found that the increase in shape, s (location of branch point in side chain) increases the thermostability, whereas, an opposite trend is observed for Gibbs free energy change of hydration for native proteins, GhN, in 14 families. A good correlation is observed between these two properties and the simultaneous increases of -GhN and s is necessary to enhance the thermostability from mesophile to thermophile. The increase in shape, which tends to increase with increasing number of carbon atoms both for polar and non-polar residues, may generate more packing and compactness, and the position of beta and higher order branches may be important for better packing. On the other hand, the increase in -GhN in thermophilic proteins increases the solubility of the proteins. This tendency counterbalances the increases in insolubility and unfolding heat capacity change due to the increase in the number of carbon atoms. Thus, the present results suggest that the stability of thermophilic proteins may be achieved by a balance between better packing and solubility.     

A modified method for amino acid analysis with ninhydrin of Coomassie-stained proteins from polyacrylamide gels

Triton X-100 (from three different suppliers) and Brij 35, substituted ethers of polyoxyethylene alcohols, were found to contain variable amounts of powerful oxidizing impurities representing a range of 0.04-0.22% H₂O₂ equivalents. These detergents contain also a considerable quantity of carbonyl compounds (0.5-2%) originating from carboxylic acids and ketones or aldehydes. Tween 20, also a polyoxyethylene detergent, and sodium dodecyl sulfate were free from oxidizing contamination. Aqueous solutions of Triton X-100 and Brij 35 (1–4%) reacted readily with SH groups of protein and nonprotein molecules as well as with Fe²⁺ ion. Both detergents were purified from the oxidizing impurities by treating aqueous solution of detergent with either NaHSO₃ or SnCl₂ followed by an extraction procedure. The present findings may clarify as well as complicate the interpretation of previous studies where these detergents were used for biological purposes, especially in enzyme and protein purifications, or when present in assay procedures that are based on the formation or consumption of reducing reagents.     

Influence of amino acid properties for discriminating outer membrane proteins at better accuracy

Discriminating outer membrane proteins (OMPs) from other folding types of globular and membrane proteins is an important task both for identifying outer membrane proteins from genomic sequences and for the successful prediction of their secondary and tertiary structures. In this work, we have analyzed the influence of physico-chemical, energetic and conformational properties of amino acid residues for discriminating outer membrane proteins using different machine learning algorithms, such as, Bayes rules, Logistic functions, Neural networks, Support vector machines, Decision trees, etc. We observed that most of the properties have discriminated the OMPs with similar accuracy. The neural network method with the property, free energy change could discriminate the OMPs from other folding types of globular and membrane proteins at the 5-fold cross-validation accuracy of 94.4% in a dataset of 1,088 proteins, which is better than that obtained with amino acid composition. The accuracy of discriminating globular proteins is 94.3% and that of transmembrane helical (TMH) proteins is 91.8%. Further, the neural network method is tested with globular proteins belonging to 30 major folding types and it could successfully exclude 99.4% of the considered 1612 non-redundant proteins. These accuracy levels are comparable to or better than other methods in the literature. We suggest that this method could be effectively used to discriminate OMPs and for detecting OMPs in genomic sequences.     

A new acid hydrolysis method for determining tryptophan in peptides and proteins

Three different methods for hydrolysis and determination of amino acid composition of peptides and proteins were compared. We found, that the method of Matsubara and Sasaki (using 6N HCl and thioglycolic acid) gives comparatively low recoveries for tryptophan, while Liu and Chang's method, using p-toluenesulfonic acid and tryptamine, is more suitable. To eliminate the difficulties of the latter method, we used mercaptoethane-sulfonic acid, which, in the concentration used, results in total hydrolysis of peptide bonds within 22 hr and gives very high tryptophan recoveries. Both sulfonic acid methods were used for hydrolysis of the pentapeptide “pentagastrine” as well as of the proteins lysozyme, cytochrome c, and chymotrypsine. Their amino acid composition was determined using an automatic amino acid analyzer. Similarly to the p-toluenesulfonic acid method, the results of our method are totally reliable only for pure peptides and proteins, though the results obtained with our method using samples containing carbohydrates are better than those of all earlier methods.