Genetic activity of base analogs in Salmonella typhimurium and Escherichia coli and Saccharomyces cerevisiae

The study of 6-N-hydroxylaminopurine (HAP) and 2-amino-6-N-hydroxylaminopurine (AHAP) activity in bacteria and the yeast was undertaken. AHAP was found to be more effective as a mutagen in bacteria and HAP--in the yeast. Mutagenic and lethal effects or analogues were independent of excision and mutagenic repair both in bacteria and the yeast. Deletion in uvrB region of Salmonella genome leads to hypersensitivity to lethal and mutagenic action of analogues. Both of the latter only cause reversions of base-substitution but not frameshift mutations. Considering the data obtained and the information from published papers, we proposed that HAP and AHAP exert their mutagenic action, like classical analogues, by means of incorporation into DNA and disturbing the regular replication laws.     

用有限酶切法分析蛋白质的氨基酸序列

报道了一个通过有限酶切蛋白质产生多肽片段的方法.蛋白质经单向SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离和用考马斯亮蓝短暂染色后,切下所需的蛋白质带,将其放入另一个SDS-PAGE凝胶的样品槽内,在电泳过程中该蛋白质被蛋白酶如蛋白酶V8降解,所产生的多肽片段随之被分离.电泳结束后,将多肽片段电印迹至聚偏二氟乙烯(polyvinylidene difluoride,PVDF)膜上.这些多肽片段从PVDF膜上切下后可以直接被用于分析氨基酸序列.该方法能广泛适用于分析一般蛋白质和N端被修饰蛋白质的氨基酸序列.     

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.     

Protein splicing: Excision of intervening sequences at the protein level

Protein splicing is an extraordinary post-translational reaction that removes an intact central “spacer” domain (Sp) from precursor proteins (N-Sp-C) while splicing together the N- and C-domains of the precursor, via a peptide bond, to produce a new protein (N-C). All of the available data on protein splicing fit a model in which these intervening sequences excise at the protein level via a self-splicing mechanism. Several proteins have recently been discovered that undergo protein splicing, and in two such cases, the excised spacer protein is an endonuclease. Such endonucleases are capable of conferring genetic mobility upon the intervening sequences that encodes them. These intervening sequences define a new family of mobile genetic elements that are translated yet remain phenotypically silent by excising at the protein rather than the RNA level.     

Rapid identification of proteins by peptide-mass fingerprinting

BACKGROUND: Developments in 'soft' ionisation techniques have revolutionized mass-spectro-metric approaches for the analysis of protein structure. For more than a decade, such techniques have been used, in conjuction with digestion b specific proteases, to produce accurate peptide molecular weight 'fingerprints' of proteins. These fingerprints have commonly been used to screen known proteins, in order to detect errors of translation, to characterize post-translational modifications and to assign diulphide bonds. However, the extent to which peptide-mass information can be used alone to identify unknown sample proteins, independent of other analytical methods such as protein sequence analysis, has remained largely unexplored. RESULTS: We report here on the development of the molecular weight search (MOWSE) peptide-mass database at the SERC Daresbury Laboratory. Practical experience has shown that sample proteins can be uniquely identified from a few as three or four experimentally determined peptide masses when these are screened against a fragment database that is derived from over 50 000 proteins. Experimental errors of a few Daltons are tolerated by the scoring algorithms, thus permitting the use of inexpensive time-of-flight mass spectrometers. As with other types of physical data, such as amino-acid composition or linear sequence, peptide masses provide a set of determinants that are sufficiently discriminating to identify or match unknown sample proteins. CONCLUSION: Peptide-mass fingerprints can prove as discriminating as linear peptide sequences, but can be obtained in a fraction of the time using less protein. In many cases, this allows for a rapid identification of a sample protein before committing it to protein sequence analysis. Fragment masses also provide information, at the protein level, that is complementary to the information provided by large-scale DNA sequencing or mapping projects.     

Analysis of secreted proteins from Aspergillus flavus

MS/MS techniques in proteomics make possible the identification of proteins from organisms with little or no genome sequence information available. Peptide sequences are obtained from tandem mass spectra by matching peptide mass and fragmentation information to protein sequence information from related organisms, including unannotated genome sequence data. This peptide identification data can then be grouped and reconstructed into protein data. In this study, we have used this approach to study protein secretion by Aspergillus flavus, a filamentous fungus for which very little genome sequence information is available. A. flavus is capable of degrading the flavonoid rutin (quercetin 3-O-glycoside), as the only source of carbon via an extracellular enzyme system. In this continuing study, a proteomic analysis was used to identify secreted proteins from A. flavus when grown on rutin. The growth media glucose and potato dextrose were used to identify differentially expressed secreted proteins. The secreted proteins were analyzed by 1- and 2-DE and MS/MS. A total of 51 unique A. flavus secreted proteins were identified from the three growth conditions. Ten proteins were unique to rutin-, five to glucose- and one to potato dextrose-grown A. flavus. Sixteen secreted proteins were common to all three media. Fourteen identifications were of hypothetical proteins or proteins of unknown functions. To our knowledge, this is the first extensive proteomic study conducted to identify the secreted proteins from a filamentous fungus.     

Selection of cryptic B-cell epitopes using informational analysis of protein sequences

Sub-unit vaccines are synthetic or recombinant peptides representing T- or B-cell epitopes of major protein antigens from a particular pathogen. Epitope selection requires the synthesis of peptides that overlap the protein sequences and screening for the most effective ones. In this study a new method of immunogenic peptide selection based on the analysis of information structure of protein sequences is suggested. The analysis of known B-cell epitope location in the information structure of Aspergillus fumigatus proteins Asp f 2 and Asp f 3 has shown that epitopes are scattered along the sequences of proteins for the exception of sites with Increased Degree Information Coordination (IDIC). Based on these results peptides from different allergens such as Asp f 2, Der p 1, and Fel d 1 were selected and produced in a recombinant form in the context of yeast virus-like particles (VLPs). Immunization of mice with VLPs containing peptides form allergens has induced the production of IgG able to recognize full-length antigens. This result suggests that the analysis of information structure of proteins can be used for the selection of peptides possessing cryptic B-cell epitope activity.     

Biosynthesis of vitamin B6: direct identification of the product of the PdxA-catalyzed oxidation of 4-hydroxy-l-threonine-4-phosphate using electrospray ionization mass spectrometry

PdxA (E.C. 1.1.1.262) catalyzes a key step in the biosynthesis of vitamin B(6): the nicotinamide-dependent oxidation of 4-hydroxy-l-threonine-4-phosphate (HTP) to a product tentatively identified as 3-amino-1-hydroxyacetone 1-phosphate (AHAP). To date, the evidence for the formation of AHAP, while self-consistent, has been largely circumstantial, and does not exclude the possibility that the actual product of the enzyme-catalyzed oxidation of HTP might be 2-amino-3-oxo-4-hydroxybutyric acid 4-phosphate which could undergo rapid, non-enzyme-catalyzed decarboxylation once released from the protein. Use of negative ion electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometric analysis (MS-MS) confirms that AHAP is the product of the PdxA-catalyzed reaction.     

Candidate Biomarker Discovery for Angiogenesis by Automatic Integration of Orbitrap MS1 Spectral-and X!Tandem MS2 Sequencing Information

Candidate protein biomarker discovery by full automatic integration of Orbitrap full MS1 spectral peptide profiling and X!Tandem MS2 peptide sequencing is investigated by analyzing mass spectra from brain tumor samples using Peptrix. Potential protein candidate biomarkers found for angiogenesis are compared with those previously reported in the literature and obtained from previous Fourier transform ion cyclotron resonance (FT-ICR) peptide profiling. Lower mass accuracy of peptide masses measured by Orbitrap compared to those measured by FT-ICR is compensated by the larger number of detected masses separated by liquid chromatography (LC), which can be directly linked to protein identifications. The number of peptide sequences divided by the number of unique sequences is 9248/6911 ≈ 1.3. Peptide sequences appear 1.3 times redundant per up-regulated protein on average in the peptide profile matrix, and do not seem always up-regulated due to tailing in LC retention time (40%), modifications (40%) and mass determination errors (20%). Significantly up-regulated proteins found by integration of X!Tandem are described in the literature as tumor markers and some are linked to angiogenesis. New potential biomarkers are found, but need to be validated independently. Eventually more proteins could be found by actively involving MS2 sequence information in the creation of the MS1 peptide profile matrix.     

De novo sequence analysis of N-terminal sulfonated peptides after in-gel guanidination

Here we report a novel approach in which gel-separated proteins are guanidinated in-gel prior to enzymatic cleavage. In contrast to previously described techniques, this procedure allows the extracted tryptic peptides to be N-terminal sulfonated without any further sample purification. The derivatized peptides were subsequently fragmented using a matrix-assisted laser desorption/ionization time of flight/time of flight instrument. The approach facilitates the de novo sequence analysis and allows obtaining longer stretches of amino acid sequence information. We demonstrate that the obtained information can be used to identify proteins using a sequence similarity search algorithm. The technique was compared to the standard peptide mass fingerprint approach, applied either in-gel or in solution, using a number of sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated model proteins. Finally, the new protocol was applied on a proteomic study of two-dimensional PAGE separated proteins from Shewanella oneidensis. More than 50 proteins from this organism were identified using sub-picomol quantities of protein, and peptide sequences of up to 20 amino acid residues in length have been determined.     

Tangible benefits of the aphid Acyrthosiphon pisum genome sequencing for aphid proteomics: Enhancements in protein identification and data validation for homology-based proteomics

Homology-driven proteomics promises to reveal functional biology in insects with sparse genome sequence information. A proteomics study comparing plant virus transmission competent and refractive genotypes of the aphid Schizaphis graminum isolated numerous candidate proteins involved in virus transmission, but limited genome sequence information hampered their identification. The complete genome of the pea aphid, Acyrthosiphon pisum, released in 2008, enabled us to double the number of protein identifications beyond what was possible using available EST libraries and other insect sequences. This was concomitant with a dramatic increase of the number of MS and MS/MS peptide spectra matching the genome-derived protein sequence. LC-MS/MS proved to be the most robust method of peptide detection. Cross-matching spectral data to multiple EST sequences and error tolerant searching to identify amino acid substitutions enhanced the percent coverage of the Schizaphis graminum proteins. 2-D electrophoresis provided the protein pI and MW which enabled the refinement of the candidate protein selection and provided a measure of protein abundance when coupled to the spectral data. Thus, the homology-based proteomics pipeline for insects should include efforts to maximize the number of peptide matches to the protein to increase certainty in protein identification and relative protein abundance.     

MALDI-TOF质谱源后衰变技术鉴定2D胶蛋白点

PMF方法由于具有高灵敏度、高通量和容易自动化等优点,在蛋白质组学鉴定中占有重要的地位。然而,许多样品(比如：小分子蛋白,混合物等)仅仅通过PMF方法不能明确鉴定。在这种情况下,在测定PMF的同一个样品上,选择一个酶解片段峰进行PSD测序,并把这些序列信息输入MS—Tag软件进行搜索,结合PMF方法,表观分子量等电点等参数,能够对胶上的点进行明确的鉴定。本文先用PSD方法对胶上的三个标准蛋白进行鉴定,都得到了非常准确的结果,同时鉴定了胶上的几个未知点。     

Arabidopsis nuclear-encoded plastid transit peptides contain multiple sequence subgroups with distinctive chloroplast-targeting sequence motifs

The N-terminal transit peptides of nuclear-encoded plastid proteins are necessary and sufficient for their import into plastids, but the information encoded by these transit peptides remains elusive, as they have a high sequence diversity and lack consensus sequences or common sequence motifs. Here, we investigated the sequence information contained in transit peptides. Hierarchical clustering on transit peptides of 208 plastid proteins showed that the transit peptide sequences are grouped to multiple sequence subgroups. We selected representative proteins from seven of these multiple subgroups and confirmed that their transit peptide sequences are highly dissimilar. Protein import experiments revealed that each protein contained transit peptide-specific sequence motifs critical for protein import into chloroplasts. Bioinformatics analysis identified sequence motifs that were conserved among members of the identified subgroups. The sequence motifs identified by the two independent approaches were nearly identical or significantly overlapped. Furthermore, the accuracy of predicting a chloroplast protein was greatly increased by grouping the transit peptides into multiple sequence subgroups. Based on these data, we propose that the transit peptides are composed of multiple sequence subgroups that contain distinctive sequence motifs for chloroplast targeting.     

Proteome analysis of wheat lemma

We report here for the first time on the construction of proteomes from wheat lemma at the anthesis stage. After transfer of lemma proteins to polyvinylidene difluoride membranes, seventy larger spots were subjected to peptide sequence analysis; the amino acid sequences could be described for forty-eight of these proteins. The result suggested that wheat proteins were less N-terminally blocked compared to rice proteins, which are known to have a much higher ratio of N-terminal blocks. We further analyzed the internal sequences of eight blocked proteins by the Cleveland peptide mapping method. Out of these total 56 amino acid sequences, forty-one could be assigned to the corresponding expressed sequence tags (ESTs). The expression profile of lemma proteins was generally similar to that of leaf, and the majority of identified proteins were related to cellular metabolisms. We analyzed the internal sequences of one protein spot present in lemma, which was not present in leaf.     

A proteomic approach to identification of transmembrane proteins and membrane-anchored proteins of Arabidopsis thaliana by peptide sequencing.

A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved effective for the identification of membrane-bound proteins.     

Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation

Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.     

Proteome analysis of the leukocytes from the American alligator (Alligator mississippiensis) using mass spectrometry

Mass spectrometry was used in conjunction with gel electrophoresis and liquid chromatography, to determine peptide sequences from American alligator (Alligator mississippiensis) leukocytes and to identify similar proteins based on homology. The goal of the study was to generate an initial database of proteins related to the alligator immune system. We have adopted a typical proteomics approach for this study. Proteins from leukocyte extracts were separated using two-dimensional gel electrophoresis and the major bands were excised, digested and analyzed by on-line nano-LC MS/MS to generate peptide sequences. The sequences generated were used to identify proteins and characterize their functions. The protein identity and characterization of the protein function were based on matching two or more peptides to the same protein by searching against the NCBI database using MASCOT and Basic Local Alignment Search Tool (BLAST). For those proteins with only one peptide matching, the phylum of the matched protein was considered. Forty-three proteins were identified that exhibit sequence similarities to proteins from other vertebrates. Proteins related to the cytoskeletal system were the most abundant proteins identified. These proteins are known to regulate cell mobility and phagocytosis. Several other peptides were matched to proteins that potentially have immune-related function.     

Identification of structured peptide segments in folding proteins.

Prediction of the structures of long polypeptides and small proteins has been carried out using conformational energy calculations. These calculations can be applied to large proteins if structured regions of their sequences can be identified. Three different approaches to identifying such sequences are presented. First, sequences of five or more contiguous hydrophobic residues tend to nucleate alpha-helices. Second, peptide sequences from parent proteins that have the same biological activities as the parent proteins are highly structured. Third, structured synthetic peptide segments from proteins inhibit the folding of the parent proteins by competing with the corresponding segment of the protein chain for associating with complementary regions. Examples of each of these approaches are presented.