Purification and characterization of carbohydrate-binding peptides from Lotus tetragonolobus and Ulex europeus seed lectins using affinity chromatography.

Carbohydrate-binding peptides of several anti-H(O) leguminous lectins were obtained from endoproteinase Asp-N or Lys-C digests of L-fucose-binding Lotus tetragonolobus lectin (LTA) and Ulex europeus lectin I (UEA-I) and from that of a di-N-acetylchitobiose-binding Ulex europeus lectin II (UEA-II) by affinity chromatography on columns of Fuc-Gel (for LTA and UEA-I) and on a column of a mixture of several oligomers of N-acetyl-D-glucosamine (GlcNAc) coupled to Sepharose 4B (GlcNAc oligomer-Sepharose 4B) (for UEA-II). These peptides were retained on the Fuc-Gel or GlcNAc oligomer-Sepharose 4B column and were presumed to have an affinity for the columns. The amino acid sequences of the retarded peptides were determined using a protein sequencer.     

Gas-phase cleavage of PTC-derivatized electrosprayed tryptic peptides in an FT-ICR trapped-ion cell: Mass-based protein identification without liquid chromatographic separation

Condensed phase protein sequencing typically relies on N-terminal labeling with phenylisothiocyanate ("Edman" reagent), followed by cleavage of the N-terminal amino acid. Similar Edman degradation has been observed in the gas phase by collision-activated dissociation of the N-terminal phenyl thiocarbamoyl protonated peptide [1] to yield complementary b1 and y(n-1) fragments, identifying the N-terminal amino acid. By use of infrared multiphoton (rather than collisional) activation, and Fourier transform ion cyclotron resonance (rather than quadrupole) mass analysis, we extend the method to direct analysis of a mixture of tryptic peptides. We validate the approach with bradykinin as a test peptide, and go on to analyze a mixture of 25 peptides produced by tryptic digestion of apomyoglobin. A b1+ ion is observed for three of the Edman-derivatized peptides, thereby identifying their N-terminal amino-acids. Search of the SWISS-PROT database gave a single hit (myoglobin, from the correct biological species), based on accurate-mass FT-ICR MS for as few as one Edman-derivatized tryptic peptide. The method is robust-it succeeds even with partial tryptic digestion, partial Edman derivatization, and partial MS/MS IRMPD cleavage. Improved efficiency and automation should be straightforward.     

Separation and characterization of proteins from green and etiolated shoots of rice (Oryza sativa L.): towards a rice proteome

Proteins extracted from green and etiolated shoots of rice were separated by two-dimensional polyacrylamide gel electrophoresis and relative molecular weights and isoelectric points were determined. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and 85 proteins were analyzed by a gas-phase protein sequencer. The N-terminal amino acid sequences of 21 out of 85 proteins were determined in this manner. N-terminal regions of the remaining proteins could not be sequenced. The internal amino acid sequences of proteins were determined by sequence analysis of peptides obtained by the Cleveland peptide mapping method and compared with those of known plant and animal protein sequences to understand the nature of the proteins. Green shoots revealed the presence of photosynthetic proteins as expected; however, as etiolated shoots were not photosynthetic, only precursors of the photosynthetic proteins were identified. Interestingly, the presence of L-ascorbate peroxidase only in etiolated shoots suggests a cellular protectant function for this antioxidant enzyme in the etiolating shoots. Using this experimental approach, we could identify the major proteins involved in growth regulation in photosynthetic green shoots as well as in etiolating rice seedlings.     

Selective isolation of N-terminal peptides from proteins and their de novo sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without regard to unblocking or blocking of N-terminal amino acids

We have developed a new method to determine the N-terminal amino acid sequences of proteins, regardless of whether their N-termini are modified. This method consists of the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling of sulfo-NHS-SS-biotin to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease followed by oxidation with performic acid; (4) specific isolation of N-terminal peptides from digests using DITC resins; (5) de novo sequence analysis of the N-terminal peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the CAF (chemically assisted fragmentation) method or tandem mass spectrometric (MS/MS) analysis according to unblocked or blocked peptides, respectively. By employing DITC resins instead of avidin resins used in our previous method (Yamaguchi et al., Rapid Commun. Mass Spectrom. 2007; 21: 3329), it has been possible to isolate selectively N-terminal peptides from proteins regardless of modification of N-terminal amino acids. Here we propose a universal method for N-terminal sequence analysis of proteins.     

The acidic domains of the Toc159 chloroplast preprotein receptor family are intrinsically disordered protein domains

Background  

The Toc159 family of proteins serve as receptors for chloroplast-destined preproteins. They directly bind to transit peptides, and exhibit preprotein substrate selectivity conferred by an unknown mechanism. The Toc159 receptors each include three domains: C-terminal membrane, central GTPase, and N-terminal acidic (A-) domains. Although the function(s) of the A-domain remains largely unknown, the amino acid sequences are most variable within these domains, suggesting they may contribute to the functional specificity of the receptors.     

Composite sequence proteomic analysis of protein biomarkers of Campylobacter coli, C. lari and C. concisus for bacterial identification

Protein biomarkers observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of three strains of Campylobacter coli, two strains of C. lari and one strain of C. concisus have been identified by 'bottom-up' proteomic techniques. The significant findings are as follows. First, the protein biomarkers identified were: PhnA-related protein, 4-oxalocrotonate tautomerase (DmpI)-related protein, NifU-like protein, cytochrome c, DNA-binding protein HU, 10 kDa chaperonin, thioredoxin, as well as several conserved hypothetical and ribosomal proteins. Second, variations in the biomarker ion m/z in MALDI-TOF-MS spectra across species and strains are the result of variations in the amino acid sequence of the protein due to non-synonymous mutations of the biomarker gene. Third, the most common post-translational modifications (PTMs) were the removal of the N-terminal methionine and N-terminal signal peptides. However, in the case of the NifU protein (an iron-sulfur cluster transport protein), post-translational cleavage occurred from the C-terminus. Fourth, only the genomes of the C. coli strain RM2228 and C. lari strain RM2100 have been sequenced; thus, proteomic identification of the proteins of the other strains in this study relied upon sequence homology to the genomic sequence of these strains as well as the genomes of sequences of other Campylobacter strains. In some cases, the determination of the full amino acid sequence of a protein biomarker from a genomically non-sequenced strain was accomplished by combining non-overlapping partial sequences from proteomic identifications of genomically-sequenced strains that were of the same species (or of a different species) to that of the non-sequenced strain. The accuracy of this composite sequence was confirmed by both MS and MS/MS. It was necessary, in some cases, to perform de novo sequencing on 'gaps' in the composite sequence that were not homologous to any genomically-sequenced strain. In order to validate the composite sequence approach, composite sequences were further confirmed by subsequent DNA sequencing of the biomarker gene. Thus, using the composite sequence approach, it was possible to determine the full amino acid sequence of an unknown protein from a genomically non-sequenced bacterial strain without the necessity of either sequencing the biomarker gene or performing full de novo MS/MS sequencing. The sequence obtained could then be used as a strain-specific biomarker for analysis by 'top-down' proteomics techniques.     

Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).     

A Systematic Investigation of Proteoforms with N-Terminal Glycine and Their Dynamics Reveals Its Impacts on Protein Stability

The N-termini of proteins can regulate their degradation, and the same protein with different N-termini may have distinct dynamics. Recently, it was found that N-terminal glycine can serve as a degron recognized by two E3 ligases, but N-terminal glycine was also reported to stabilize proteins. Here we developed a chemoenzymatic method for selective enrichment of proteoforms with N-terminal glycine and integrated dual protease cleavage to further improve the enrichment specificity. Over 2000 unique peptides with protein N-terminal glycine were analyzed from >1000 proteins, and most of them are previously unknown, indicating the effectiveness of the current method to capture low-abundance proteoforms with N-terminal glycine. The degradation rates of proteoforms with N-terminal glycine were quantified along with those of proteins from the whole proteome. Bioinformatic analyses reveal that proteoforms with N-terminal glycine with the fastest and slowest degradation rates have different functions and localizations. Membrane proteins with N-terminal glycine and proteins with N-terminal glycine from the N-terminal methionine excision degrade more rapidly. Furthermore, the secondary structures, adjacent amino acid residues, and protease specificities for N-terminal glycine are also vital for protein degradation. The results advance our understanding of the effects of N-terminal glycine on protein properties and functions.     

Characterization of modification sites during peptide synthesis using liquid secondary ion/collision-induced dissociation mass spectrometry and a computer program

Side-reactions often occur during peptide synthesis resulting in modified amino acid moieties. To identify these residues, liquid secondary ion/collision-induced dissociation mass spectra were recorded. The main fragments are generated by cleavage of the peptide bond. To facilitate interpretation of the spectra and assignment of the structure, a simple, but flexible and efficient, computer program is presented. The program allows the verification of the correct structure of the synthesized peptides and the deduction of the type of side-products formed, such as alkylation of tryptophan residues.     

Sequence dependent fragmentation of peptides generated by MALDI quadrupole time-of-flight (MALDI Q-TOF) mass spectrometry and its implications for protein identification

A study has been undertaken to evaluate the usefulness of MALDI Q-TOF data for protein identification. The comparison of MS data of protein digests obtained on a conventional MALDI TOF instrument to the MS data from the MALDI Q-TOF reveal peptide patterns with similar intensity ratios. However, comparison of MS/MS Q-TOF data produced by nanoelectrospray versus MALDI reveals striking differences. Peptide fragment ions obtained from doubly charged precursors produced by nanoelectrospray are mainly y-type ions with some b-ions in the lower mass range. In contrast, peptide fragment ions produced from the singly charged ions originating from the MALDI source are a mixture of y-, b- and a-ions accompanied by ions resulting from neutral loss of ammonia or water. The ratio and intensity of these fragment ions is found to be strongly sequence dependent for MALDI generated ions. The singly charged peptides generated by MALDI show a preferential cleavage of the C-terminal bond of acidic residues aspartic and glutamic acid and the N-terminal bond of proline. This preferential cleavage can be explained by the mobile proton model and is present in peptides that contain both arginine and an acidic amino acid. The MALDI Q-TOF MS/MS data of 24 out of 26 proteolytic peptides produced by trypsin or Asp-N digestions were successfully used for protein identification via database searching, thus indicating the general usefulness of the data for protein identification. De novo sequencing using a mixture of 160/18O water during digestion has been explored and de novo sequences for a number of peptides have been obtained.     

An algorithm for interpretation of low-energy collision-induced dissociation product ion spectra for de novo sequencing of peptides

An algorithm for interpretation of product ion spectra of peptides generated from ion trap mass spectrometry is developed for de novo amino acid sequencing of peptides for the purpose of protein identification. It is based on a multi-pass analysis of product ion data using a rigorous data extraction and sequence interpretation protocol in the initial pass. The extraction/interpretation algorithm becomes more relaxed in subsequent passes, considering more of the fragment ions, and potentially more sequence candidates. The possible peptide sequences generated by the algorithm are scored according to those sequences which best explain the fragment ion spectrum. These sequences are searched against a protein database using a BLAST search engine to find likely protein candidates. The method is also suitable for locating and determining protein modifications, and can be applied to de novo interpretation of peptide fragment ions in the tandem mass (MS/MS) spectrum produced from a mixture of two peptides having similar nominal mass, but different sequences. Using a known protein, bovine serum albumin, as an example, it is illustrated that this method is rapid and efficient for MS/MS spectral interpretation. This method combined with BLAST programs is then applied to search homologies and to generate information on post-translational modifications of an unknown protein isolated from shark cartilage that does not have a complete genome or proteome database.     

Comparative investigation of the molecular structures of the species-specific proteins of the seeds ofGossypium hirsutum andG. barbadense

The N-terminal amino acids and the amino acid compositions of four species-specific proteins of two species of the cotton plant —Gossypium hirsutum andG. barbadense — have been determined. Peptide maps of tryptic hydrolysates of these proteins have been obtained and compared. Individual tryptic peptides have been isolated and purified. The N-ends, amino acid compositions, and amino acid sequences of individual peptides have been determined. The N-terminal sections of the proteins ofG. hirsutum andG. barbadense as far as the 16th amino acid residue have been sequenced. A comparative analysis has been made of the peptides and proteins of these cottonplant species.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 40 64 75. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 858–864, November–December, 1997.     

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.     

The correct molecular weight of myoglobin, a common calibrant for mass spectrometry.

Myoglobins from horse heart muscle, horse skeletal muscle and sperm whale are widely used as calibration standards or test compounds for various mass spectrometric methodologies. In all such cases reported in the literature, a molecular weight value is used (16,950.5 and 17,199, respectively) which is based on the assumption that amino acid 122 in this 153 amino-acid-long protein is asparagine, overlooking a published suggestion that it is aspartic acid instead. Since the mass assignment accuracy for matrix-assisted laser desorption mass spectrometry is reported to be +/- 0.01% and for electrospray ionization +/- 0.0025%, and error of one mass unit in approximately 17,000 would be significant. The mass-to-charge ratio of ions of the tryptic peptide encompassing amino acid 122 derived from commercially available horse heart and horse skeletal myoglobins, the apomyoglobin of the latter, and the tryptic and chymotryptic peptide of sperm whale myoglobin proved that in both proteins amino acid 122 is indeed aspartic acid, rather than asparagine. This finding was further confirmed by the collision-induced dissociation spectra of the [M + H]+ ions of the tryptic peptides from the horse myoglobins and the chymotriptic peptide from sperm whale myoglobin. Thus, the correct molecular weight of horse myoglobin is 16,951.49 and that of the sperm whale protein is 17,199.91.     

Studies on the primary structure of cow kappa-casein. Structural features of para-kappa-casein; N-terminal sequence of kappa-caseinoglycopeptide studied with a sequencer

Several long tryptic peptides obtained from reduced maleylated χ-casein were sequenced: they belonged to the N- and C-terminal (37 residues) moieties of para-χ-casein. Several tryptic peptides could then be joined by chymotryptic overlap peptides. 102 amino acid residues of para-χ-casein were thus placed into two long and four shorter sequences. Some structural results were established with a Sequencer which was also employed for a verification of the N-terminal sequence of the χ-caseinoglycopeptide; a previously described short glycopeptide was found again and was reinvestigated.     

Selective N-terminal modification of peptides and proteins: Recent progresses and applications

Numerous strategies for linking desired chemical probes with target peptides and proteins have been developed and applied in the field of biological chemistry. Approaches for site-specific modification of native amino acid residues in test tubes and biological contexts represent novel biological tools for understanding the role of peptides and proteins. Selective N-terminal modification strategies have been broadly studied especially in the last 10 years, as N-terminal positions are typically so...     

Conformation Analysis of Ile-Ala-Val-Pro Peptide and Its Derivatives by Circular Dichroism

Ile-Ala-Val-Pro as a hypocholesterolemic peptide was isolated from soybean protein. We have synthesized four peptides, Ile-Ala-Val-Pro-Gly-Glu-Val-Ala, Leu-Ile-Ala-Val-Pro-Gly-Glu-Val-Ala, Ile-Ala-Val-Pro-Thr-Gly-Val-Ala, Leu-Ile-Ala-Val-Pro-Thr-Gly-Val-Ala, with a conserved Ile-Ala-Val-Pro amino acid sequence, for circular dichroism investigations. These four peptide sequences were also found in the amino acid sequence in soybean protein, which was defined from the genomic sequence. Additionally for a detailed analysis of conformation features of these peptides, the Ile-Ala-Val-Pro and Leu-Ile-Ala-Val-Pro were also synthesized. All peptides were prepared using standard fluorenylmethyloxycarbonyl methodology and the peptide yields ranged from 90 to 95% of the theoretical yields with purity after purification above 99%.     

Peptide mobility and peptide mapping in capillary zone electrophoresis. Experimental determination and theoretical simulation.

The electrophoretic mobilities of 58 peptides that varied in size from 2 to 39 amino acids and varied in charge from 0.65 to 7.82 are presented. The measurements were conducted at 22 degrees C using a 10% linear polyacrylamide-coated column and a 50 mM phosphate buffer at pH 2.5. Excellent separation of peptides and highly reliable peptide maps of protein digests are routinely obtained using these experimental conditions. The electrophoretic data were used to test existing theoretical models that correlate electrophoretic mobility with physical parameters. The results indicate that the Offord model that correlates electrophoretic mobility with the charge-to-size parameter q/M2/3 offers the best fit of our reliable experimental data. Furthermore, we also obtained the capillary zone electrophoretic profile of the endoproteinase Lys-C digests of a peptide sequencing standard, melittin, and horse myoglobin under the same experimental conditions as described above. The resulting peptide maps were compared with corresponding theoretical simulation.     

Tunable Charge Tags for Electron-Based Methods of Peptide Sequencing: Design and Applications

Charge tags using basic auxiliary functional groups 6-aminoquinolinylcarboxamido, 4-aminopyrimidyl-1-methylcarboxamido, 2-aminobenzoimidazolyl-1-methylcarboxamido, and the fixed-charge 4-(dimethylamino)pyridyl-1-carboxamido moiety are evaluated as to their properties in electron transfer dissociation mass spectra of arginine C-terminated peptides. The neutral tags have proton affinities that are competitive with those of amino acid residues in peptides. Charge reduction by electron transfer from fluoranthene anion-radicals results in peptide backbone dissociations that improve sequence coverage by providing extensive series of N-terminal c-type fragments without impeding the formation of C-terminal z fragments. Comparison of ETD mass spectra of free and tagged peptides allows one to resolve ambiguities in fragment ion assignment through mass shifts of c ions. Simple chemical procedures are reported for N-terminal tagging of Arg-containing tryptic peptides.