Sequence homology in the metalloproteins; purple acid phosphatase from beef spleen and uteroferrin from porcine uterus

The primary structures of purple acid phosphatase and uteroferrin, two iron-binding glycoproteins isolated from beef spleen and porcine uterine fluids, respectively, have been examined by a combination of tandem mass spectrometry and classical Edman sequencing methods. Reported here are amino acid sequence data covering more than 90% of the primary structures for these two proteins. The sequence data reveal an unexpectedly high degree of homology, greater than 90%, for these two proteins.     

RNA‐directed DNA methylation efficiency depends on trigger and target sequence identity

RNA‐directed DNA methylation (RdDM) in plants has been extensively studied, but the RNA molecules guiding the RdDM machinery to their targets are still to be characterized. It is unclear whether these molecules require full complementarity with their target. In this study, we have generated Nicotiana tabacum (Nt) plants carrying an infectious tomato apical stunt viroid (TASVd) transgene (Nt‐TASVd) and a non‐infectious potato spindle tuber viroid (PSTVd) transgene (Nt‐SB2). The two viroid sequences exhibit 81% sequence identity. Nt‐TASVd and Nt‐SB2 plants were genetically crossed. In the progeny plants (Nt‐SB2/TASVd), deep sequencing of small RNAs (sRNAs) showed that TASVd infection was associated with the accumulation of abundant small interfering RNAs (siRNAs) that mapped along the entire TASVd but only partially matched the SB2 transgene. TASVd siRNAs efficiently targeted SB2 RNA for degradation, but no transitivity was detectable. Bisulfite sequencing in the Nt‐SB2/TASVd plants revealed that the TASVd transgene was targeted for dense cis‐RdDM along its entire sequence. In the same plants, the SB2 transgene was targeted for trans‐RdDM. The SB2 methylation pattern, however, was weak and heterogeneous, pointing to a positive correlation between trigger–target sequence identity and RdDM efficiency. Importantly, trans‐RdDM on SB2 was also detected at sites where no homologous siRNAs were detected. Our data indicate that RdDM efficiency depends on the trigger–target sequence identity, and is not restricted to siRNA occupancy. These findings support recent data suggesting that RNAs with sizes longer than 24 nt (>24‐nt RNAs) trigger RdDM.     

Sequence analysis of peptide mixtures by automated integration of Edman and mass spectrometric data.

A computer algorithm is described that utilizes both Edman and mass spectrometric data for simultaneous determination of the amino acid sequences of several peptides in a mixture. Gas phase sequencing of a peptide mixture results in a list of observed amino acids for each cycle of Edman degradation, which by itself may not be informative and typically requires reanalysis following additional chromatographic steps. Tandem mass spectrometry, on the other hand, has a proven ability to analyze sequences of peptides present in mixtures. However, mass spectrometric data may lack a complete set of sequence-defining fragment ions, so that more than one possible sequence may account for the observed fragment ions. A combination of the two types of data reduces the ambiguity inherent in each. The algorithm first utilizes the Edman data to determine all hypothetical sequences with a calculated mass equal to the observed mass of one of the peptides present in the mixture. These sequences are then assigned figures of merit according to how well each of them accounts for the fragment ions in the tandem mass spectrum of that peptide. The program was tested on tryptic and chymotryptic peptides from hen lysozyme, and the results are compared with those of another computer program that uses only mass spectral data for peptide sequencing. In order to assess the utility of this method the program is tested using simulated mixtures of varying complexity and tandem mass spectra of varying quality.     

The Nt17 Domain and its Helical Conformation Regulate the Aggregation,Cellular Properties and Neurotoxicity of Mutant Huntingtin Exon 1

Converging evidence points to the N-terminal domain comprising the first 17 amino acids of the Huntingtin protein (Nt17) as a key regulator of its aggregation, cellular properties and toxicity. In this study, we further investigated the interplay between Nt17 and the polyQ domain repeat length in regulating the aggregation and inclusion formation of exon 1 of the Huntingtin protein (Httex1). In addition, we investigated the effect of removing Nt17 or modulating its local structure on the membrane interactions, neuronal uptake, and toxicity of monomeric or fibrillar Httex1. Our results show that the polyQ and Nt17 domains synergistically modulate the aggregation propensity of Httex1 and that the Nt17 domain plays important roles in shaping the surface properties of mutant Httex1 fibrils and regulating their poly-Q-dependent growth, lateral association and neuronal uptake. Removal of Nt17 or disruption of its transient helical conformations slowed the aggregation of monomeric Httex1 in vitro, reduced inclusion formation in cells, enhanced the neuronal uptake and nuclear accumulation of monomeric Httex1 proteins, and was sufficient to prevent cell death induced by Httex1 72Q overexpression. Finally, we demonstrate that the uptake of Httex1 fibrils into primary neurons and the resulting toxicity are strongly influenced by mutations and phosphorylation events that influence the local helical propensity of Nt17. Altogether, our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation, internalization, and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, and neuronal toxicity.     

Modulation of Polyglutamine Conformations and Dimer Formation by the N-Terminus of Huntingtin

Polyglutamine expansions within different proteins are associated with nine different neurodegenerative diseases. There is growing interest in understanding the roles of flanking sequences from disease-relevant proteins in the intrinsic conformational and aggregation properties of polyglutamine. We report results from atomistic simulations and circular dichroism experiments that quantify the effect of the N-terminal 17-residue (Nt17) segment of the huntingtin protein on polyglutamine conformations and intermolecular interactions. We show that the Nt17 segment and polyglutamine domains become increasingly disordered as polyglutamine length (N) increases in Nt17-Q_N constructs. Hydrophobic groups within Nt17 become sequestered in intramolecular interdomain interfaces. We also show that the Nt17 segment suppresses the intrinsic propensity of polyglutamine aggregation. This inhibition arises from the incipient micellar structures adopted by monomeric forms of the peptides with Nt17 segments. The degree of intermolecular association increases with increasing polyglutamine length and is governed mainly by associations between polyglutamine domains. Comparative analysis of intermolecular associations for different polyglutamine-containing constructs leads to clearer interpretations of recently published experimental data. Our results suggest a framework for fibril formation and identify roles for flanking sequences in the modulation of polyglutamine aggregation.     

Sequence analysis of bovine lens aldose reductase

The covalent structure of bovine lens aldose reductase (alditol-NADP+ oxidoreductase, EC 1.1.1.21) was determined by sequence analysis of peptides generated by specific and chemical cleavage of the homogeneous apoenzyme. Peptides, purified by reverse-phase high performance liquid chromatography were subjected to compositional analysis and sequencing by gas-phase automated Edman degradation. Aldose reductase was found to contain 315 amino acid residues. The enzyme is blocked at the amino terminus, and mass spectrometry was employed to identify the blocking acetyl group and to sequence the amino-terminal tryptic peptide. The aldose reductase was shown to contain no carbohydrate despite the fact that the enzyme contains the consensus sequence -Asn-Lys-Thr- for N-linked glycosylation. Comparative sequence analysis and application of algorithms for prediction of secondary structure and nucleotide binding domains are consistent with the view that aldose reductase is a double-domain protein with a beta-alpha-beta secondary structural organization. The NADPH binding site appears to be associated with the amino-terminal half of the enzyme. Modeling studies based on the tertiary structures of dihydrofolate and glutathione reductases indicate that the NADPH binding site begins at Lys-11 and continues with a beta-alpha-beta fold characteristic of nucleotide binding proteins.     

Sequence Analysis of a Proteolyzed Site in Drosophila Choline Acetyltransferase

The amino terminal sequence of the 13-kilodalton (kD) polypeptide present in purified Drosophila acetyl-CoA: choline-O-acetyltransferase (EC 2.3.1.6) was determined, and its position in the sequence of the intact enzyme was located. Enzyme polypeptides for sequencing were obtained from native enzyme protein by denaturation, followed by fractionation on reverse-phase HPLC. The 13-, 54-, and 67-kD polypeptides recovered from the separation were subjected to amino terminal sequencing. Only the 13-kD fragment yielded a sequence. The 67- and 54-kD polypeptides appeared completely blocked to gas-phase Edman sequencing. The location of the amino terminal sequence from the 13-kD polypeptide in the cDNA-deduced enzyme sequence indicated that this fragment represents the carboxyl portion of the 67-kD enzyme, with the 54-kD polypeptide providing the amino terminal portion. The proteolysis that gave rise to the 13-kD polypeptide occurred at the carboxyl side of a monobasic lysine residue. An earlier comparison of the enzyme from Drosophila and pigs indicated that the cleaved lysine may be a conserved residue in the porcine enzyme. The cleaved enzyme region characterized in this study does not coincide with the regions of high homology found in the two enzymes, but hydrophilicity profiles generated for this area showed similarities.     

The ABRF Edman Sequencing Research Group 2008 Study: Investigation into Homopolymeric Amino Acid N-Terminal Sequence Tags and Their Effects on Automated Edman Degradation

The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.     

A proteomic analysis of leaf sheaths from rice

The proteins extracted from the leaf sheaths of rice seedlings were separated by 2-D PAGE, and analyzed by Edman sequencing and mass spectrometry, followed by database searching. Image analysis revealed 352 protein spots on 2-D PAGE after staining with Coomassie Brilliant Blue. The amino acid sequences of 44 of 84 proteins were determined; for 31 of these proteins, a clear function could be assigned, whereas for 12 proteins, no function could be assigned. Forty proteins did not yield amino acid sequence information, because they were N-terminally blocked, or the obtained sequences were too short and/or did not give unambiguous results. Fifty-nine proteins were analyzed by mass spectrometry; all of these proteins were identified by matching to the protein database. The amino acid sequences of 19 of 27 proteins analyzed by mass spectrometry were similar to the results of Edman sequencing. These results suggest that 2-D PAGE combined with Edman sequencing and mass spectrometry analysis can be effectively used to identify plant proteins.     

A strategy for the rapid identification of phosphorylation sites in the phosphoproteome

Edman phosphate ((32)P) release sequencing provides a high sensitivity means of identifying phosphorylation sites in proteins that complements mass spectrometry techniques. We have developed a bioinformatic assessment tool, the cleavage of radiolabeled protein (CRP) program, which enables experimental identification of phosphorylation sites via (32)P labeling and Edman degradation of cleaved proteins obtained at femtomole levels. By observing the Edman cycle(s) in which radioactivity is found, candidate phosphorylation sites are identified by determining which residues occur at the observed number of cycles downstream from a peptide cleavage site. In cases where more than one residue could be responsible for the observed radioactivity, additional experiments with cleavage reagents having alternative specificities may resolve the ambiguity. Given a protein sequence and a cleavage site, CRP performs these experiments in silico, identifying resolved sites based on user-supplied experimental data, as well as suggesting combinations of reagents for additional analyses. Analysis of the PhosphoBase protein sequence database suggests that CRP data from two cleavage experiments can be used to identify unambiguously 60% of known phosphorylation sites. Data from additional cleavage experiments may increase the overall coverage to 70% of known sites. By comparing theoretical data obtained from the CRP program with (32)P release data obtained from an Edman sequencer, a known phosphorylation site was identified unambiguously and correctly. In addition, our results show that in vivo phosphorylation sites can be determined routinely by differential proteolysis analysis and Edman cycling with less than 1 fmol of protein and 1000 cpm.     

CRP: Cleavage of Radiolabeled Phosphoproteins

The CRP (Cleavage of Radiolabeled Phosphoproteins) program guides the design and interpretation of experiments to identify protein phosphorylation sites by Edman sequencing of unseparated peptides. Traditionally, phosphorylation sites are determined by cleaving the phosphoprotein and separating the peptides for Edman 32P-phosphate release sequencing. CRP analysis of a phosphoprotein's sequence accelerates this process by omitting the separation step: given a protein sequence of interest, the CRP program performs an in silico proteolytic cleavage of the sequence and reports the predicted Edman cycles in which radioactivity would be observed if a given serine, threonine or tyrosine were phosphorylated. Experimentally observed cycles containing 32P can be compared with CRP predictions to confirm candidate sites and/or explore the ability of additional cleavage experiments to resolve remaining ambiguities. To reduce ambiguity, the phosphorylated residue (P-Tyr, P-Ser or P-Thr) can be determined experimentally, and CRP will ignore sites with alternative residues. CRP also provides simple predictions of likely phosphorylation sites using known kinase recognition motifs. The CRP interface is available at http://fasta.bioch.virginia.edu/crp.     

Proteome Analysis of Light-induced Proteins in Synechocystis sp. PCC 6803: Identification of Proteins Separated by 2D-PAGE Using N-terminal Sequencing and MALDI-TOF MS

The cyanobacterium Synechocystis sp. PCC 6803 is an ideal model organism for the proteome study of light-induced gene expression because the whole genomic sequence has been determined. The soluble proteins extracted from light- and dark-cultured cells were separated by two-dimensional polyacrylamide gel electrophoresis. Light-induced protein spots electroblotted on a polyvinyldiene difluoride membrane were analyzed by N-terminal Edman sequence determination and followed by CyanoBase. The tryptic digests of some proteins were also confirmed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and MS-Fit search. Interestingly, eight proteins were related to photosynthesis and respiration (RbcS/L, CbbA, Gap2, AtpB, CpcB, PsbO, and PsbU). Four proteins (SodB, DnaK, GroEL2, and Tig) were involved in cellular processes and the functions of another two proteins (rehydrin and membrane protein) were unknown. The proteome analysis by N-terminal Edman sequencing and MALDI-TOF enabled us to characterize one-shot protein profiles expressed under different physiological conditions.     

串联质谱在多肽测序中的应用

采用纳升喷雾(Nano)技术和碰撞诱导解离(CID collision inducd dissociation)方法,在电喷雾-四极杆-飞行时间质谱(ESI-Q-TOF electrspectrometry ionization-quadrupole-time of flight)上,对两种序列部分未知的天然多肽进行从头测序(de novo sequence),结果证明质谱的de novo sequence可以方便有效的解决传统的Edman降解法测序中常见的实际问题,如末位残基的丢失,赖氨酸和亮氨酸难鉴定等,此方法的建立是对Edman降解测序法很好的补充．     

A new strategy for primary structure determination of proteins: application to bovine beta-casein

A new approach has been developed for sequencing proteins. A radioactive label is attached specifically to the C-terminus of the protein. The labelled molecule is subjected to varying proteolysis conditions. From the electrophoretic patterns (SDS-PAGE) of the hydrolysates, appropriate cleavage conditions are selected, giving labelled peptides of different lengths which are purified. The labelled peptides are sequenced in order of increasing size (from 1 to n), peptide (i) being sequenced until the N-terminal sequence of peptide (i-1) is encountered. This approach allows the determination of a complete protein sequence with a minimal number of Edman cycles. The method was successfully applied to bovine beta-casein (209 residues) which was completely resequenced with only 239 Edman cycles.     

大麦条纹花叶病毒新疆株(BSMV-XJ)RNA2 3′端结构分析

本文利用双脱氧序列分析法对我国大麦条纹花叶病毒新疆株(BSMV-XJ)RNA2 cDNA的3′端进行序列分析,证明XJ株RNA2 3′端239个核苷酸与国外典型株3′端相应部位有高度的序列同源性。通过序列分析及使用寡核苷酸定位裂解法和分子杂交确定,在紧邻239个核苷酸的上游有一个Poly(A)结构,3′终端为一个类tRNA结构,亦与国外典型株相同。经分析认为BSMV-XJ3个基因组RNA具有相同的3′端结构。     

Apolipophorin-III-like protein expressed in the antenna of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae)

Antennal proteins of the male fire ant (Solenopsis invicta) were analyzed by two-dimensional gel electrophoresis, with the objective of identifying pheromone-binding proteins, which have not previously been found in ant antennae. The major low-molecular weight protein found in the male fire ant antenna was subjected to Edman degradation to determine the N-terminal amino acid sequence. Degenerate PCR primers based on this sequence were used to obtain a cDNA sequence corresponding to the full-length protein sequence. In-gel trypsin digestion followed by MALDI-TOF mass spectrometry and HPLC-ESI/MS/MS demonstrated that the protein gel spot contained only the protein corresponding to the cDNA sequence obtained by PCR. The sequence is similar to apolipophorin-III, an exchangeable lipid-binding protein. Fire ant apolipophorin-III is expressed in the antenna as well as the head, thorax and abdomen.     

Protein sequence analysis using Hewlett-Packard biphasic sequencing cartridges in an applied biosystems 473A protein sequencer

Protein sequence analysis using an adsorptive biphasic sequencing cartridge, a set of two coupled columns introduced by Hewlett-Packard for protein sequencing by Edman degradation, in an Applied Biosystems 473A protein sequencer has been demonstrated. Samples containing salts, detergents, excipients, etc. (e.g., formulated protein drugs) can be easily analyzed using the ABI sequencer. Simple modifications to the ABI sequencer to accommodate the cartridge extend its utility in the analysis of difficult samples. The ABI sequencer solvents and reagents were compatible with the HP cartridge for sequencing. Sequence information up to ten residues can be easily generated by this nonoptimized procedure, and it is sufficient for identifying proteins by database search and for preparing a DNA probe for cloning novel proteins.     

In situ cyanogen bromide cleavage of N-terminally blocked proteins in a gas-phase sequencer

Herein we describe a procedure for the in situ cyanogen bromide cleavage of N-terminally blocked proteins which have been immobilised onto the glass fiber sample disk of the gas-phase sequencer. In this manner, new amino terminii suitable for automated Edman degradation can be generated. Cytochrome C was cleaved using this method on the carboxyl side of methionine residues 65 and 80. This allowed sequence analysis to begin simultaneously at residues 66 and 81 (Table 1). This procedure offers an alternative sequencing tactic for methionine-containing proteins which are N-terminally blocked.     

Sequence of the gene for ribosomal protein L23 from the archaebacterium Methanococcus vannielii

The N-terminal sequence of HPLC-purified protein L23 from the Methanococcus vannielii ribosome has been determined by automated liquid-phase Edman degradation. Using the N-terminal amino acid sequence, an oligonucleotide probe complementary to the 5'-end of the gene was synthesized. The 26-mer oligonucleotide, containing two inosines, was used for hybridization with digested M. vannielii chromosomal DNA. The hybridizing band from HpaII-digested genomic DNA was ligated into pUC18 to yield plasmid pMvaZ1 containing the entire gene of protein L23. The nucleotide sequence complemented the partial amino acid sequence, and the gene codes for a protein of 9824 Da. The amino acid sequence of protein L23 form M. vannielii was compared to that of ribosomal proteins from other archaebacteria as well as from eubacteria and eukaryotes. The number of identical amino acids is highest when the M. vannielii protein is compared to the homologous protein from yeast and lowest vs that from tobacco chloroplasts. Interestingly, the secondary structures of the proteins as predicted by computer programs are more conserved than the primary structures.