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52-517: Entrez Global Query is an integrated search and retrieval system that provides access to all databases simultaneously with a single query string and user interface. Entrez can efficiently retrieve related sequences , structures , and references. The Entrez system can provide views of gene and protein sequences and chromosome maps. Some textbooks are also available online through the Entrez system. The Entrez front page provides, by default, access to

104-618: A MyNCBI account can save queries indefinitely, and also choose to have updates with new search results e-mailed for saved queries of most databases. It is widely used in the field of biotechnology as a reference tool for students and professionals alike. Entrez searches the following databases: In addition to using the search engine forms to query the data in Entrez, NCBI provides the Entrez Programming Utilities (eUtils) for more direct access to query results. The eUtils are accessed by posting specially formed URLs to

156-478: A hydroxyoxazolidine (Ser/Thr) or hydroxythiazolidine (Cys) intermediate]. This intermediate tends to revert to the amide form, expelling the attacking group, since the amide form is usually favored by free energy, (presumably due to the strong resonance stabilization of the peptide group). However, additional molecular interactions may render the amide form less stable; the amino group is expelled instead, resulting in an ester (Ser/Thr) or thioester (Cys) bond in place of

208-415: A primary structure, although the usage is not standard. The primary structure of a biological polymer to a large extent determines the three-dimensional shape ( tertiary structure ). Protein sequence can be used to predict local features , such as segments of secondary structure, or trans-membrane regions. However, the complexity of protein folding currently prohibits predicting the tertiary structure of

260-479: A protease of different specificity may also be useful. Whilst detailed comparison of the MS data with predictions based on the known protein sequence may be used to define post-translational modifications, targeted approaches to data acquisition may also be used. For instance, specific enrichment of phosphopeptides may assist in identifying phosphorylation sites in a protein. Alternative methods of peptide fragmentation in

312-555: A protein by the Edman degradation follows; some of the steps are elaborated on subsequently. Peptides longer than about 50–70 amino acids long cannot be sequenced reliably by the Edman degradation. Because of this, long protein chains need to be broken up into small fragments that can then be sequenced individually. Digestion is done either by endopeptidases such as trypsin or pepsin or by chemical reagents such as cyanogen bromide . Different enzymes give different cleavage patterns, and

364-494: A protein from its sequence alone. Knowing the structure of a similar homologous sequence (for example a member of the same protein family ) allows highly accurate prediction of the tertiary structure by homology modeling . If the full-length protein sequence is available, it is possible to estimate its general biophysical properties , such as its isoelectric point . Sequence families are often determined by sequence clustering , and structural genomics projects aim to produce

416-514: A reagent that will form a coloured derivative. If the amounts of amino acids are in excess of 10 nmol, ninhydrin can be used for this; it gives a yellow colour when reacted with proline, and a vivid purple with other amino acids. The concentration of amino acid is proportional to the absorbance of the resulting solution. With very small quantities, down to 10 pmol, fluorescent derivatives can be formed using reagents such as ortho-phthaldehyde (OPA) or fluorescamine . Pre-column derivatization may use

468-537: A set of representative structures to cover the sequence space of possible non-redundant sequences. Peptide sequencing Protein sequencing is the practical process of determining the amino acid sequence of all or part of a protein or peptide . This may serve to identify the protein or characterize its post-translational modifications . Typically, partial sequencing of a protein provides sufficient information (one or more sequence tags) to identify it with reference to databases of protein sequences derived from

520-407: A similar interface for searching each particular database and for refining search results. The Limits feature allows the user to narrow a search, a web forms interface. The History feature gives a numbered list of recently performed queries. Results of previous queries can be referred to by number and combined via Boolean operators. Search results can be saved temporarily in a Clipboard. Users with

572-421: A variety of reagents to prevent or reduce degradation, such as thiol reagents or phenol to protect tryptophan and tyrosine from attack by chlorine, and pre-oxidising cysteine. He also suggests measuring the quantity of ammonia evolved to determine the extent of amide hydrolysis . The amino acids can be separated by ion-exchange chromatography then derivatized to facilitate their detection. More commonly,

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624-416: Is a machine that performs Edman degradation in an automated manner. A sample of the protein or peptide is immobilized in the reaction vessel of the protein sequenator and the Edman degradation is performed. Each cycle releases and derivatises one amino acid from the protein or peptide's N -terminus and the released amino-acid derivative is then identified by HPLC. The sequencing process is done repetitively for

676-584: Is as follows: Hydrolysis is done by heating a sample of the protein in 6 M hydrochloric acid to 100–110 °C for 24 hours or longer. Proteins with many bulky hydrophobic groups may require longer heating periods. However, these conditions are so vigorous that some amino acids ( serine , threonine , tyrosine , tryptophan , glutamine , and cysteine ) are degraded. To circumvent this problem, Biochemistry Online suggests heating separate samples for different times, analysing each resulting solution, and extrapolating back to zero hydrolysis time. Rastall suggests

728-647: Is generally not useful to determine the positions of disulfide bridges. It also requires peptide amounts of 1 picomole or above for discernible results, making it less sensitive than mass spectrometry . In biology, proteins are produced by translation of messenger RNA (mRNA) with the protein sequence deriving from the sequence of codons in the mRNA. The mRNA is itself formed by the transcription of genes and may be further modified. These processes are sufficiently understood to use computer algorithms to automate predictions of protein sequences from DNA sequences, such as from whole-genome DNA-sequencing projects, and have led to

780-413: Is harder because of the reverse information loss (from amino acids to DNA sequence). The current lossless data compressor that provides higher compression is AC2. AC2 mixes various context models using Neural Networks and encodes the data using arithmetic encoding. The proposal that proteins were linear chains of α-amino acids was made nearly simultaneously by two scientists at the same conference in 1902,

832-440: Is much smaller than the number of available methods of N-terminal analysis. The most common method is to add carboxypeptidases to a solution of the protein, take samples at regular intervals, and determine the terminal amino acid by analysing a plot of amino acid concentrations against time. This method will be very useful in the case of polypeptides and protein-blocked N termini. C-terminal sequencing would greatly help in verifying

884-444: Is often sufficient to confirm the termini (thus that the protein’s measured mass matches that predicted from its sequence) and infer the presence or absence of many post-translational modifications. Proteolysis does not always yield a set of readily analyzable peptides covering the entire sequence of POI. The fragmentation of peptides in the mass spectrometer often does not yield ions corresponding to cleavage at each peptide bond. Thus,

936-434: The cyclol hypothesis advanced by Dorothy Wrinch , proposed that the linear polypeptide underwent a chemical cyclol rearrangement C=O + HN → {\displaystyle \rightarrow } C(OH)-N that crosslinked its backbone amide groups, forming a two-dimensional fabric . Other primary structures of proteins were proposed by various researchers, such as the diketopiperazine model of Emil Abderhalden and

988-406: The active site of the protein, inhibiting its function. The protein is activated by cleaving off the inhibitory peptide. Some proteins even have the power to cleave themselves. Typically, the hydroxyl group of a serine (rarely, threonine) or the thiol group of a cysteine residue will attack the carbonyl carbon of the preceding peptide bond, forming a tetrahedrally bonded intermediate [classified as

1040-516: The amino -terminal end through to the carboxyl -terminal end. Either a three letter code or single letter code can be used to represent the 22 naturally encoded amino acids, as well as mixtures or ambiguous amino acids (similar to nucleic acid notation ). Peptides can be directly sequenced , or inferred from DNA sequences . Large sequence databases now exist that collate known protein sequences. In general, polypeptides are unbranched polymers, so their primary structure can often be specified by

1092-521: The encoded 22, and may be cyclised, modified and cross-linked. Peptides can be synthesised chemically via a range of laboratory methods. Chemical methods typically synthesise peptides in the opposite order (starting at the C-terminus) to biological protein synthesis (starting at the N-terminus). Protein sequence is typically notated as a string of letters, listing the amino acids starting at

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1144-575: The protein has been synthesized on the ribosome , typically occurring in the endoplasmic reticulum , a subcellular organelle of the eukaryotic cell. Many other chemical reactions (e.g., cyanylation) have been applied to proteins by chemists, although they are not found in biological systems. In addition to those listed above, the most important modification of primary structure is peptide cleavage (by chemical hydrolysis or by proteases ). Proteins are often synthesized in an inactive precursor form; typically, an N-terminal or C-terminal segment blocks

1196-405: The pyrrol/piperidine model of Troensegaard in 1942. Although never given much credence, these alternative models were finally disproved when Frederick Sanger successfully sequenced insulin and by the crystallographic determination of myoglobin and hemoglobin by Max Perutz and John Kendrew . Any linear-chain heteropolymer can be said to have a "primary structure" by analogy to the usage of

1248-528: The 1920s when he argued that rubber was composed of macromolecules . Thus, several alternative hypotheses arose. The colloidal protein hypothesis stated that proteins were colloidal assemblies of smaller molecules. This hypothesis was disproved in the 1920s by ultracentrifugation measurements by Theodor Svedberg that showed that proteins had a well-defined, reproducible molecular weight and by electrophoretic measurements by Arne Tiselius that indicated that proteins were single molecules. A second hypothesis,

1300-523: The 74th meeting of the Society of German Scientists and Physicians, held in Karlsbad. Franz Hofmeister made the proposal in the morning, based on his observations of the biuret reaction in proteins. Hofmeister was followed a few hours later by Emil Fischer , who had amassed a wealth of chemical details supporting the peptide-bond model. For completeness, the proposal that proteins contained amide linkages

1352-811: The DNA sequences of their genes. Further protein characterization may include confirmation of the actual N- and C-termini of the POI, determination of sequence variants and identification of any post-translational modifications present. A general scheme for protein identification is described. The pattern of fragmentation of a peptide allows for direct determination of its sequence by de novo sequencing . This sequence may be used to match databases of protein sequences or to investigate post-translational or chemical modifications. It may provide additional evidence for protein identifications performed as above. The peptides matched during protein identification do not necessarily include

1404-495: The Edman reagent to produce a derivative that is detected by UV light. Greater sensitivity is achieved using a reagent that generates a fluorescent derivative. The derivatized amino acids are subjected to reversed phase chromatography, typically using a C8 or C18 silica column and an optimised elution gradient. The eluting amino acids are detected using a UV or fluorescence detector and the peak areas compared with those for derivatised standards in order to quantify each amino acid in

1456-496: The N- or C-termini predicted for the matched protein. This may result from the N- or C-terminal peptides being difficult to identify by MS (e.g. being either too short or too long), being post-translationally modified (e.g. N-terminal acetylation) or genuinely differing from the prediction. Post-translational modifications or truncated termini may be identified by closer examination of the data (i.e. de novo sequencing). A repeat digest using

1508-679: The NCBI server, and parsing the XML response. There was also an eUtils SOAP interface which was terminated in July 2015. In 1991, Entrez was introduced in CD form. In 1993, a client-server version of the software provided connectivity with the internet. In 1994, NCBI established a website, and Entrez was a part of this initial release. In 2001, Entrez bookshelf was released and in 2003, the Entrez Gene database

1560-494: The amine group of the N-terminal amino acid. The terminal amino acid can then be selectively detached by the addition of anhydrous acid. The derivative then isomerises to give a substituted phenylthiohydantoin , which can be washed off and identified by chromatography, and the cycle can be repeated. The efficiency of each step is about 98%, which allows about 50 amino acids to be reliably determined. A protein sequenator

1612-519: The amino acids are derivatized then resolved by reversed phase HPLC . An example of the ion-exchange chromatography is given by the NTRC using sulfonated polystyrene as a matrix, adding the amino acids in acid solution and passing a buffer of steadily increasing pH through the column. Amino acids are eluted when the pH reaches their respective isoelectric points . Once the amino acids have been separated, their respective quantities are determined by adding

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1664-414: The cloned DNA was then determined and used to deduce the full amino-acid sequence of the protein. Bioinformatics tools exist to assist with interpretation of mass spectra (see de novo peptide sequencing ), to compare or analyze protein sequences (see sequence analysis ), or search databases using peptide or protein sequences (see BLAST ). The difficulty of protein sequencing was recently proposed as

1716-405: The conceptual translation of genes . The two major direct methods of protein sequencing are mass spectrometry and Edman degradation using a protein sequenator (sequencer). Mass spectrometry methods are now the most widely used for protein sequencing and identification but Edman degradation remains a valuable tool for characterizing a protein's N -terminus . It is often desirable to know

1768-463: The deduced sequence for each peptide is not necessarily complete. The standard methods of fragmentation do not distinguish between leucine and isoleucine residues since they are isomeric. Because the Edman degradation proceeds from the N-terminus of the protein, it will not work if the N-terminus has been chemically modified (e.g. by acetylation or formation of Pyroglutamic acid). Edman degradation

1820-618: The determination of amino acid composition, with the exception that no stain is needed, as the reagents produce coloured derivatives and only qualitative analysis is required. So the amino acid does not have to be eluted from the chromatography column, just compared with a standard. Another consideration to take into account is that, since any amine groups will have reacted with the labelling reagent, ion exchange chromatography cannot be used, and thin-layer chromatography or high-pressure liquid chromatography should be used instead. The number of methods available for C-terminal amino acid analysis

1872-442: The generation of large databases of protein sequences such as UniProt . Predicted protein sequences are an important resource for protein identification by mass spectrometry. Historically, short protein sequences (10 to 15 residues) determined by Edman degradation were back-translated into DNA sequences that could be used as probes or primers to isolate molecular clones of the corresponding gene or complementary DNA. The sequence of

1924-415: The global query. All databases indexed by Entrez can be searched via a single query string, supporting Boolean operators and search term tags to limit parts of the search statement to particular fields. This returns a unified results page, that shows the number of hits for the search in each of the databases, which are also linked to actual search results for that particular database. Entrez also provides

1976-455: The laboratory. Protein primary structures can be directly sequenced , or inferred from DNA sequences . Amino acids are polymerised via peptide bonds to form a long backbone , with the different amino acid side chains protruding along it. In biological systems, proteins are produced during translation by a cell's ribosomes . Some organisms can also make short peptides by non-ribosomal peptide synthesis , which often use amino acids other than

2028-464: The mass spectrometer, such as ETD or ECD , may give complementary sequence information. The protein’s whole mass is the sum of the masses of its amino-acid residues plus the mass of a water molecule and adjusted for any post-translational modifications. Although proteins ionize less well than the peptides derived from them, a protein in solution may be able to be subjected to ESI-MS and its mass measured to an accuracy of 1 part in 20,000 or better. This

2080-400: The overlap between fragments can be used to construct an overall sequence. The peptide to be sequenced is adsorbed onto a solid surface. One common substrate is glass fibre coated with polybrene , a cationic polymer . The Edman reagent, phenylisothiocyanate (PITC), is added to the adsorbed peptide, together with a mildly basic buffer solution of 12% trimethylamine . This reacts with

2132-407: The peptide bond. This chemical reaction is called an N-O acyl shift . The ester/thioester bond can be resolved in several ways: The compression of amino acid sequences is a comparatively challenging task. The existing specialized amino acid sequence compressors are low compared with that of DNA sequence compressors, mainly because of the characteristics of the data. For example, modeling inversions

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2184-480: The primary structures of proteins predicted from DNA sequences and to detect any posttranslational processing of gene products from known codon sequences. The Edman degradation is a very important reaction for protein sequencing, because it allows the ordered amino acid composition of a protein to be discovered. Automated Edman sequencers are now in widespread use, and are able to sequence peptides up to approximately 50 amino acids long. A reaction scheme for sequencing

2236-438: The protein can undergo a variety of post-translational modifications , which are briefly summarized here. The N-terminal amino group of a polypeptide can be modified covalently, e.g., The C-terminal carboxylate group of a polypeptide can also be modified, e.g., Finally, the peptide side chains can also be modified covalently, e.g., Most of the polypeptide modifications listed above occur post-translationally , i.e., after

2288-604: The sample. Determining which amino acid forms the N -terminus of a peptide chain is useful for two reasons: to aid the ordering of individual peptide fragments' sequences into a whole chain, and because the first round of Edman degradation is often contaminated by impurities and therefore does not give an accurate determination of the N -terminal amino acid. A generalised method for N -terminal amino acid analysis follows: There are many different reagents which can be used to label terminal amino acids. They all react with amine groups and will therefore also bind to amine groups in

2340-420: The sequence of amino acids along their backbone. However, proteins can become cross-linked, most commonly by disulfide bonds , and the primary structure also requires specifying the cross-linking atoms, e.g., specifying the cysteines involved in the protein's disulfide bonds. Other crosslinks include desmosine . The chiral centers of a polypeptide chain can undergo racemization . Although it does not change

2392-428: The sequence, it does affect the chemical properties of the sequence. In particular, the L -amino acids normally found in proteins can spontaneously isomerize at the C α {\displaystyle \mathrm {C^{\alpha }} } atom to form D -amino acids, which cannot be cleaved by most proteases . Additionally, proline can form stable trans-isomers at the peptide bond. Additionally,

2444-418: The side chains of amino acids such as lysine - for this reason it is necessary to be careful in interpreting chromatograms to ensure that the right spot is chosen. Two of the more common reagents are Sanger's reagent ( 1-fluoro-2,4-dinitrobenzene ) and dansyl derivatives such as dansyl chloride . Phenylisothiocyanate , the reagent for the Edman degradation, can also be used. The same questions apply here as in

2496-468: The term for proteins, but this usage is rare compared to the extremely common usage in reference to proteins. In RNA , which also has extensive secondary structure , the linear chain of bases is generally just referred to as the "sequence" as it is in DNA (which usually forms a linear double helix with little secondary structure). Other biological polymers such as polysaccharides can also be considered to have

2548-591: The unordered amino acid composition of a protein prior to attempting to find the ordered sequence, as this knowledge can be used to facilitate the discovery of errors in the sequencing process or to distinguish between ambiguous results. Knowledge of the frequency of certain amino acids may also be used to choose which protease to use for digestion of the protein. The misincorporation of low levels of non-standard amino acids (e.g. norleucine) into proteins may also be determined. A generalized method often referred to as amino acid analysis for determining amino acid frequency

2600-423: The whole polypeptide until the entire measurable sequence is established or for a pre-determined number of cycles. Protein identification is the process of assigning a name to a protein of interest (POI), based on its amino-acid sequence. Typically, only part of the protein’s sequence needs to be determined experimentally in order to identify the protein with reference to databases of protein sequences deduced from

2652-400: Was developed. Primary structure Protein primary structure is the linear sequence of amino acids in a peptide or protein . By convention, the primary structure of a protein is reported starting from the amino -terminal (N) end to the carboxyl -terminal (C) end. Protein biosynthesis is most commonly performed by ribosomes in cells. Peptides can also be synthesized in

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2704-574: Was made as early as 1882 by the French chemist E. Grimaux. Despite these data and later evidence that proteolytically digested proteins yielded only oligopeptides, the idea that proteins were linear, unbranched polymers of amino acids was not accepted immediately. Some scientists such as William Astbury doubted that covalent bonds were strong enough to hold such long molecules together; they feared that thermal agitations would shake such long molecules asunder. Hermann Staudinger faced similar prejudices in

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