Direct microsequence analysis of polypeptides using an improved sequenator, a nonprotein carrier (polybrene), and high pressure liquid chromatography

We have combined the use of a nonprotein carrier (Polybrene), high pressure liquid chromatography, and modifications in Edman chemistry with the improvements of a commercial spinning cup sequenator suggested by Wittmann-Liebold [Wittmann-Liebold, B. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 1415] to analyze amino acid phenylthiohydantoins obtained from automated Edman degradation of microquantities of polypeptide directly without the use of radiolabel. This approach has allowed us to determine the sequence of the N-terminal 47 residues of sperm whale myoglobin starting with 200 pmol of protein, 77 residues of an antibody light chain with 5 nmole of protein, and 54 residues of an antibody heavy chain with 8 nmol of protein. In addition, we completely sequenced a hydrophobic 14-residue peptide at the 1.5-nmol level. Our technique of direct analysis for microsamples is capable of providing routine, extende N-terminal sequence analysis for nanomole and subnanomole levels of polypeptides and protines, and it also is applicable to analysis of more classical sample quantities.     

Automated microsequence analysis by use of radioactive phenylisothiocyanate.

The use of radioactive phenylisothiocyanate as a coupling reagent in conjunction with an automated protein sequenator permits N-terminal sequence analysis of 1.5 nmoles of protein. This procedure should facilitate chemical studies on several membrane proteins of current interest (e.g., histocompatability and Ir-associated alloantigens) which are available in only small quantities.     

Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machines based on n-peptide compositions

Gram-negative bacteria have five major subcellular localization sites: the cytoplasm, the periplasm, the inner membrane, the outer membrane, and the extracellular space. The subcellular location of a protein can provide valuable information about its function. With the rapid increase of sequenced genomic data, the need for an automated and accurate tool to predict subcellular localization becomes increasingly important. We present an approach to predict subcellular localization for Gram-negative bacteria. This method uses the support vector machines trained by multiple feature vectors based on n-peptide compositions. For a standard data set comprising 1443 proteins, the overall prediction accuracy reaches 89%, which, to the best of our knowledge, is the highest prediction rate ever reported. Our prediction is 14% higher than that of the recently developed multimodular PSORT-B. Because of its simplicity, this approach can be easily extended to other organisms and should be a useful tool for the high-throughput and large-scale analysis of proteomic and genomic data.     

Biopharmaceutical production: Applications of surface plasmon resonance biosensors

Surface plasmon resonance (SPR) permits the quantitative analysis of therapeutic antibody concentrations and impurities including bacteria, Protein A, Protein G and small molecule ligands leached from chromatography media. The use of surface plasmon resonance has gained popularity within the biopharmaceutical industry due to the automated, label free, real time interaction that may be exploited when using this method. The application areas to assess protein interactions and develop analytical methods for biopharmaceutical downstream process development, quality control, and in-process monitoring are reviewed.     

Manual gas-phase isothiocyanate degradation

We describe a manual gas-phase isothiocyanate degradation procedure for the primary structure determination of proteins and peptides. The proteins and peptides are applied to a polybrene-coated glass fiber filter wedged into a small glass column. The phenylisothiocyanate is directly pipetted onto the filter disk. The coupling and cleavage reactions are performed in small desiccators containing trimethylamine and trifluoroacetic acid vapors, respectively. The wash and extraction steps are performed by allowing the suitable solvents to percolate through the filter disk. The extracted anilinothiazolinone is then converted to the phenylthiohydantoin and identified by any one of a number of described methods. Our results show that this method is very sensitive and that the reactions proceed faster than those of the published automated procedure. No expensive equipment is required and the manual degradation can be performed by a laboratory assistant. A large number of samples can be simultaneously subjected to the degradation under identical conditions, making this an ideal method for physicochemical investigations into the isothiocyanate degradation. We also use this method to screen HPLC fractions after enzymatic protein fragmentation. Manually sequenced glass filters can be transferred to the automated instrument for more extended degradations.     

Separate genes encode functionally equivalent ADP/ATP carrier proteins in Saccharomyces cerevisiae. Isolation and analysis of AAC2

Genetic and biochemical analysis of Saccharomyces cerevisiae containing a disruption of the nuclear gene (AAC1) encoding the mitochondrial ADP/ATP carrier has revealed a second gene for this protein. The second gene, designated AAC2, has been isolated by genetic complementation and sequenced. AAC2 contains a 954-base pair open reading frame coding for a protein of 318 amino acids which is highly homologous to the AAC1 gene product except that it is nine amino acids longer at the NH2 terminus. The two yeast genes are highly conserved at the level of DNA and protein and share identity with the ADP/ATP carriers from other organisms. Both genes complement an ADP/ATP carrier defect (op1 or pet9). However, the newly isolated gene AAC2 need be present only in one or two copies while the previously isolated AAC1 gene must be present in multiple copies to support growth dependent on a functional carrier protein. This gene dosage-dependent complementation combined with the high degree of conservation suggest that these two functionally equivalent genes may be differentially expressed.     

Characterization of chromatin domains by 3D fluorescence microscopy: An automated methodology for quantitative analysis and nuclei screening

Fluorescence microscopy has provided a route to qualitatively analyze features of nuclear structures and chromatin domains with increasing resolution. However, it is becoming increasingly important to develop tools for quantitative analysis. Here, we present an automated method to quantitatively determine the enrichment of several endogenous factors, immunostained in pericentric heterochromatin domains in mouse cells. We show that this method permits an unbiased characterization of changes in the enrichment of several factors with statistical significance from a large number of nuclei. Furthermore, the nuclei can be sorted according to the enrichment value of these factors. This method should prove useful to monitor events related to changes in the amount, rather than the presence or absence, of any factor. By adapting a few parameters, it could be extended to other nuclear structures and the benefit of using available software will permit its use in many biological labs.     

Phloem loading of sucrose: Update and opportunities in molecular biology

Sucrose accumulates in the phloem against a concentration gradient via a presumed sucrose-specific carrier protein located at the plasmalemma of the sieve elements/companion cells. Recent evidence suggests that sucrose carrier in soybean is a 62-kDa protein. Immunocytochemical localization has shown the protein to be exclusively at the plasmalemma, which is also the site of sucrose transport. To enhance our understanding of the phenomenon, the structural gene of the sucrose carrier must be cloned and sequenced. Furthermore, development of appropriate probes should help answer long-standing questions relative to the molecular nature of sugar transport and phloem loading, the mechanism of induction/activation of sugar carriers, and developmental regulation of expression of genes encoding such carriers.     

Amino acid sequence determination of protein biomarkers of Campylobacter upsaliensis and C. helveticus by "composite" sequence proteomic analysis

We have identified the protein biomarkers observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of five strains of Campylobacter upsaliensis and one strain of C. helveticus by "bottom-up" proteomic techniques. Only one C. upsaliensis strain had previously been genomically sequenced. The significant findings are as follows: (1) The protein biomarkers identified were: 10 kD chaperonin, protein of unknown function (DUF465), phnA protein, probable periplasmic protein, D-methionine-binding lipoprotein MetQ, cytochrome c family protein, DNA-binding protein HU, thioredoxin, asparigenase family protein, helix-turn-helix domain protein, as well as several ribosomal and conserved hypothetical proteins. (2) Amino acid substitutions in protein biomarkers across species and strains account for variations in biomarker ion mass-to-charge (m/z). (3) The most common post-translational modifications (PTMs) identified were cleavage of N-terminal methionine and N-terminal signal peptides. The rule that predicts N-terminal methionine cleavage, based on the penultimate residue, does not appear to apply to C. upsaliensis proteins when the penultimate residue is threonine. (4) It was discovered that some protein biomarker genes of the genomically sequenced C. upsaliensis strain were found to have nucleotide sequences with GTG or TTG "start" codons that were not the actual start codon (ATG) of the protein based on proteomic analysis. (5) Proteomic identification of the protein biomarkers of the non-genomically sequenced C. upsaliensis and C. helveticus strains involved identification of homologous protein amino acid sequences to that of the sequenced strain. Interestingly, some protein sequence regions that were not completely homologous to the sequenced strain, due to amino acid substitutions, were found to have homologous sequence regions from more phyogenetically distant species/strains, e.g., C. jejuni. Exploiting this partial homology of more distant species/strains, it was possible to construct a "composite" amino acid sequence using multiple non-overlapping sequence regions from both phylogenetically proximate and distant strains. The new composite sequence was confirmed by both MS and MS/MS data. Thus, it was possible in some cases to determine the amino acid sequence of an unknown protein biomarker from a genomically non-sequenced bacterial strain without the necessity of either genetically sequencing the biomarker gene or resorting to de novo MS/MS analysis of the full protein sequence.     

The amino acid sequence of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B

We have determined the primary structure of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B. The enzyme is a dimeric protein of two identical subunits, each consisting of a polypeptide chain of 131 residues and a Mr = 14,536. The intact S-carboxymethyl protein was sequenced from the NH2 terminus using standard automated Edman degradation and automated Edman degradation using fluorescamine treatment at known prolines to suppress background. The isomerase was fragmented using CNBr, trypsin, iodosobenzoic acid, and acid cleavage at aspartyl-prolyl peptide bonds. The peptides resulting from each fragmentation were separated by reversed-phase high performance liquid chromatography and sequenced by automated Edman degradation. The full sequence was deduced by the overlapping of the various peptides. A search for homologous proteins was performed. Only the oxosteroid isomerase from Pseudomonas testosteroni, an expected homology, was found to be similar. Comparison of the two proteins shows that the region of strongest homology is the region containing the aspartic acid at which steroidal affinity and photoaffinity reagents have been shown to react in the P. testosteroni isomerase. The P. putida isomerase contains 3 cysteines and 2 tryptophans, whereas the P. testosteroni isomerase lacks these amino acids. The two proteins are not highly conserved.     

Functional staging of ADP/ATP carrier translocation across the outer mitochondrial membrane.

The ADP/ATP carrier (AAC) is the major representative of the inner membrane carrier proteins of mitochondria that are synthesized without cleavable presequences. The characterization of the import pathway of AAC into mitochondria has mainly depended on an operational staging system. Here, we introduce two approaches for analyzing the import of AAC, blue native electrophoresis and folding-induced translocation arrest, that allow a functional staging of AAC transport across the outer membrane. (i) Blue native electrophoresis permits a direct monitoring of the receptor stage of AAC and its chase into mitochondria. Binding to this stage requires the receptor protein Tom70 but not Tom37 or Tom20. (ii) A fusion protein between AAC and dihydrofolate reductase can be selectively arrested in the general import pore complex of the outer membrane by ligand induced folding of the passenger protein. Cross-linking demonstrates that the arrested preprotein is in close contact not only with several receptors and Tim10 but also with the channel protein Tom40, providing the first direct evidence that cleavable preproteins and carrier preproteins interact with the same outer membrane channel. The staging system presented here permits a molecular dissection of AAC transport across the outer mitochondrial membrane, relates it to functional units of the translocases, and indicates a coordinated and successive cooperation of distinct translocase subcomplexes during transfer of the preprotein.     

Protein function annotation by homology-based inference

With many genomes now sequenced, computational annotation methods to characterize genes and proteins from their sequence are increasingly important. The BioSapiens Network has developed tools to address all stages of this process, and here we review progress in the automated prediction of protein function based on protein sequence and structure.     

VistaClara: an expression browser plug-in for Cytoscape

SUMMARY: VistaClara is a plug-in for Cytoscape which provides a more flexible means to visualize gene and protein expression within a network context. An extended attribute browser is provided in the form of a graphical and interactive permutation matrix that resembles the heat map displays popular in gene-expression analysis. This extended browser permits a variety of display options and interactions not currently available in Cytoscape. AVAILABILITY: http://chianti.ucsd.edu/cyto_web/plugins/index.php.     

国产“水山姜”的分类学研究—来自核糖体DNA ITS区序列的证据

用直接测序法对国产黑果山姜Alpinia nigra(Gaertn.)Burtt以及“水山姜Alpinia aquatica (Koen.)Rose”。的核糖体DNA中的内转录间隔区（ITS）序列进行了测定,结果显示两者序列完全一致;ITS1长度为178bp,ITS2长度为232bp,5.8S编码区长度为164bp,GC含量为56.9%,形态学特征结合DNA分子证据,认为《中国植物志》记载的水山姜实为黑果山姜。     

Database-independent, database-dependent, and extended interpretation of peptide mass spectra in VEMS V2.0

The Virtual Expert Mass Spectrometrist (VEMS) program package was developed for flexible, automated, and manual de novo tandem mass spectrometry (MS/MS) protein sequencing, and includes accessory programs for matrix-assisted laser desorption/ionization-mass spectrometry (MS) interpretation, and generation of protein and peptide databases. VEMS V2.0 has been developed into a fast tool for combining database-independent and -dependent protein assignments in an extended analysis of MS/MS-peptide data. MS or MS/MS data can be directly recalibrated after the first search by fitting the data to the best search result using polynomial equations. The score function is an improvement of known scoring algorithms and can be adapted for any MS instrument type. In addition, VEMS offers a novel statistical model for evaluating the significance of the protein assignment. The novel features are illustrated by the analysis of the fragmentation spectra obtained by liquid chromatrography-MS/MS analysis of peptides from an anionic peroxidase enriched protein fraction from potato root tissue. The extended analysis mode resulted in the additional assignment of spectra for nine modified tryptic peptides and nine miscleaved peptides, in addition to the 45 spectra from regular tryptic peptides. Of the nine modified peptides, three were glycosylated.     

The amino acid sequence of rat liver non-specific lipid transfer protein (sterol carrier protein 2) is present in a high molecular weight protein: evidence from cDNA analysis

The affinity-purified antibody against rat liver non-specific lipid transfer protein (nsL-TP; sterol carrier protein 2) was used to screen a lambda-gt11 rat liver cDNA library. Positive cDNA clones were further identified by Southern blot analysis and sequenced. The largest cDNA clone consisted of 1851 bp starting at the 5' end with an open reading frame of 1545 bp. The 369 bp located at the 3' end of this open reading frame corresponded with the amino acid sequence of nsL-TP.     

Canine RPGRIP1 mutation establishes cone-rod dystrophy in miniature longhaired dachshunds as a homologue of human Leber congenital amaurosis

Cone-rod dystrophy 1 (cord1) is a recessive condition that occurs naturally in miniature longhaired dachshunds (MLHDs). We mapped the cord1 locus to a region of canine chromosome CFA15 that is syntenic with a region of human chromosome 14 (HSA14q11.2) containing the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) gene. Mutations in RPGRIP1 have been shown to cause Leber congenital amaurosis, a group of retinal dystrophies that represent the most common genetic causes of congenital visual impairment in infants and children. Using the newly available canine genome sequence we sequenced RPGRIP1 in affected and carrier MLHDs and identified a 44-nucleotide insertion in exon 2 that alters the reading frame and introduces a premature stop codon. All affected and carrier dogs within an extended inbred pedigree were homozygous and heterozygous, respectively, for the mutation. We conclude the mutation is responsible for cord1 and demonstrate that this canine disease is a valuable model for exploring disease mechanisms and potential therapies for human Leber congenital amaurosis.     

Determination of the primary structure of Eco-R1-fragment No. 5 of the rat tyrosine aminotransferase gene in the "Applied Biosystems" 370 automatic sequencer]

EcoRI-fragment No. 5 of the rat tyrosine aminotransferase gene containing exons K, L, intron 11, and a part of the 3'-nontranslatable region was digested with several restriction endonucleases (BspRI, Sau3A, BamHI, Ecl136II, AluI), the subfragments obtained were cloned into M13mp19 and sequenced using the Sanger technique with dye-labelled primers on the automated sequencer "Applied Biosystems", model 370A. The sequences were combined by means of a PC-GENE package and original programs to yield the primary structure (1064 b. p.) of the above fragment No. 5, adjacent to the previously sequenced EcoRI-fragment No. 4 (3677 b.p.).