Improvement in HPLC fractionation of thyroxine-containing thyroglobulin tryptic peptides by prior Accell ion-exchange column chromatography

The isolation and purification of the peptides containing the hormonogenic tyrosyl residues in thyroglobulin is of great interest to the understanding of structure-function relationships in this iodoprotein. This is usually performed in reduced alkylated selectively hydrolyzed thyroglobulin by subsequent HPLC fractionation. However, the main difficulty encountered when trying to isolate these peptides is their disproportion with respect to the total number of possible peptides (14 vs a total of 508). Several HPLC runs with different mobile phases are necessary and each run is accompanied with significant losses of the peptides to be purified. In an attempt to improve the separation of the T4-containing peptides in thyroglobulin tryptic digests from the much more abundant iodotyrosine-containing ones, which are present as contaminants, we have used a very simple and fast step prior to the HPLC fractionation as it is a self-packed ion-exchange column chromatography. This preliminary step results in an improvement in the separation of these peptides and leads to a relative enrichment of the hormonogenic peptides falling in different zones of the HPLC chromatogram, which facilitates their subsequent separation and purification by HPLC.     

Characterization of phosphate residues on thyroglobulin

Follicular 19 S thyroglobulin (molecular weight 660,000) from rat, human, and bovine thyroid tissues contains approximately 10-12 mol of phosphate/mol of protein. These phosphate residues can be radiolabeled when rat thyroid hemilobes, FRTL-5 rat thyroid cells, or bovine thyroid slices are incubated in vitro with [32P]phosphate. Thus labeled, the [32P]phosphate residues comigrate with unlabeled 19 S follicular thyroglobulin on sucrose gradients and gel filtration columns; are specifically immunoprecipitated by an antibody preparation to rat or bovine thyroglobulin as appropriate; and co-migrate with authentic 19 S thyroglobulin when subjected to analytic or preparative gel electrophoresis. Tunicamycin prevents approximately 50% of the phosphate from being incorporated into FRTL-5 cell thyroglobulin. Approximately one-half of the phosphate in FRTL-5 cell or bovine thyroglobulin can also be released by enzymatic deglycosylation and can be located in Pronase-digested peptides which contain mannose, are endo-beta-N-acetylglucosaminidase H but not neuraminidase-sensitive, and release a dually labeled oligosaccharide containing mannose and phosphate after endo-beta-N-acetylglucosaminidase H digestion. The remainder of the phosphate is in alkali-sensitive phosphoserine residues (3-4/mol of protein) and phosphotyrosine residues (approximately 2/mol of protein). This is evidenced by electrophoresis of acid hydrolysates of 32P-labeled thyroglobulin and by reactivity with antibodies directed against phosphotyrosine residues. The phosphoserine and phosphotyrosine residues do not appear to be randomly located through the thyroglobulin molecule since approximately 75-85% of the phosphotyrosine and phosphoserine residues were recovered in a approximately 15-kDa tryptic peptide or a approximately 24-kDa cyanogen bromide peptide, each almost devoid of carbohydrate. 31P nuclear magnetic resonance studies of bovine thyroglobulin confirm the presence and heterogeneity of the phosphate residues on thyroglobulin preparations.     

A novel method for selective isolation of C-terminal peptides from tryptic digests of proteins by immobilized anhydrotrypsin: application to structural analyses of the tail sheath and tube proteins from bacteriophage T4

A novel method useful for selective isolation of the C-terminal peptide from a tryptic digestion mixture of a protein has been developed by taking advantage of a unique property of anhydrotrypsin, which has a strong specific affinity for the peptides containing arginine or lysine at their C-termini. Briefly, peptides produced by tryptic digestion of a protein are fractionated by affinity chromatography on a column of immobilized anhydrotrypsin. The C-terminal peptide is recovered in a breakthrough fraction, while the remainders are adsorbed on the column (unless the protein ends in arginine or lysine). The breakthrough fraction is then subjected to reversed-phase high-performance liquid chromatography in order to purify the C-terminal peptide. Using this method, we have successfully isolated the C-terminal peptides from tryptic digests of the sheath protein (gp 18) and the tube protein (gp 19) of bacteriophage T4. The analytical results on these peptides, together with the information on the N-terminal structures of the original proteins and on the nucleotide sequences of genes 18 and 19, allowed us to establish the complete primary structures of the two proteins.     

Characterization and sequencing of a 40-amino-acid peptide from human thyroglobulin inducing experimental autoimmune thyroiditis.

We previously demonstrated that: a) a cytotoxic T cell hybridoma (HTC2) was able to induce lysis of syngeneic macrophages pulsed with either porcine thyroglobulin (pTg) or the tryptic fragments (TF) from pTg less than 10 kDa (M(r)) and that b) these low M(r) pTg TF included pathogenic epitopes because their injection into CBA/J mice induces thyroid lymphocytic infiltration typical of experimental autoimmune thyroiditis. Therefore the biochemical analysis of the TF preparation from pTg less than 10 kDa M(r) was undertaken and the characterized peptides were tested for their ability to be recognized or not by HTC2 cells. The sequencing of the selected peptides showed a 70% sequence homology with a portion of human thyroglobulin (hTg). The lack of a published sequence of pTg led us to synthesize a 40-amino acid peptide (F40D) similar to that portion of hTg. This F40D peptide was able to generate lymphocytic infiltrations in CBA/J mice thyroid glands, as was the native pTg molecule. Although the lymphocytic infiltrations were similar in the pTg or F40D-immunized mice, auto-antibodies to pTg or to hTg were only detectable in mice immunized with pTg. In contrast, autoantibodies levels to F40D peptide were significantly increased in serum from mice in which EAT had been induced by the F40D peptide. This highly hydrophobic peptide shows a M(r) of 4,492 kDa; it is located at the end of the second-third of the thyroglobulin molecule and up to now represents a unique sequence from the hTg molecule inducing experimental autoimmune thyroiditis.     

The proteomic analysis improved by cleavage kinetics‐based fractionation of tryptic peptides

Selective enrichment of specific peptides is an effective way to identify low abundance proteins. Fractionation of peptides prior to mass spectrometry is another widely used approach to reduce sample complexity in order to improve proteome coverage.In this study, we designed a multi‐stage digestion strategy to generate peptides with different trypsin cleavage kinetics. It was found that each of the collected peptide fractions yielded many new protein identifications compared to the control group due to the reduced complexity. The overlapping peptides identified between adjacent fractions were very low, indicating that each fraction had different sets of peptides. The multi‐stage digestion strategy separates tryptic peptides with different cleavage kinetics while RPLC separates peptides with different hydrophobicity. These two separation strategies were highly orthogonal, and showed an effective multidimensional separation to improve proteome coverage.     

Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis

The sample fractionation steps conducted prior to mass detection are critically important for the comprehensive analysis of complex protein mixtures. This paper illustrates the effectiveness of OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator for the fractionation of peptides. An Escherichia coli tryptic digest was separated in 24 fractions, and peptides were identified by reversed-phase liquid chromatography on a microfluidic device with mass spectrometric detection. About 90% of the identified individual peptides were found in only one or two fractions. The distribution of the calculated isoelectric points for the peptides identified in each fraction was especially narrow in the acidic pH range. Standard deviations approached the size of the pH segment covered by the respective fraction. The experimental peptide isoelectric point measured by OFFGEL electrophoresis was used as an additional filter for validation of peptide identifications.     

Isolation and fractionation of glycopeptides from porcine thyroglobulin

1. Glycopeptides were isolated by gel filtration on Sephadex G-25 and Sephadex G-50 from a Pronase digest of porcine thyroglobulin. 2. Isolated glycopeptides were separated into five main fractions on a column of DEAE-Sephadex A-25. Of these fractions I to III were further purified by SE-Sephadex C-25 or DEAE-Sephadex A-25 column chromatography. Several of the purified glycopeptides were homogeneous on paper electrophoresis. 3. Based on the chemical composition and molecular weight of the fractionated glycopeptides, two distinct types of heterosaccharide chain were demonstrated. 4. One type of the heterosaccharide unit consisted of four to eight residues of mannose and two residues of glucosamine and had a molecular weight of 1000-1700. The other type of unit contained sialic acid, fucose and galactose in addition to mannose and glucosamine and had a molecular weight of about 3600. 5. Mild alkaline treatment of the glycopeptide did not result in the destruction of threonine and serine. 2-Acetamido-1-N-(4-l-aspartyl)-2-deoxy-beta-d-glucopyranosylamine was isolated from partial acid hydrolysates.     

The methyl-accepting chemotaxis proteins of Escherichia coli. Identification of the multiple methylation sites on methyl-accepting chemotaxis protein I

The methyl-accepting chemotaxis proteins (MCPs) are integral membrane proteins that undergo reversible methylation during adaptation of bacterial cells to environmental attractants and repellents. The numerous methylated forms of each MCP are seen as a pattern of multiple bands on polyacrylamide gels. We have characterized the methylation sites in MCPI by analyzing methyl-accepting tryptic peptides. At least two different tryptic peptides accept methyl esters; one methyl-accepting peptide contains methionine and lysine and may be methylated a maximum of four times. The second methyl-accepting tryptic peptide contains arginine and may be methylated twice. Base-catalyzed demethylations of tryptic peptides and analysis of the charge differences between the different methylated forms of MCPI show that MCPI molecules may be methylated a total of six times. The two methyl esters on the methyl-accepting arginine peptide appear to be preferentially methylated in most of the forms of MCPI in attractant-stimulated cells. The ability to acquire six methylations on MCPI allows the bacterial cells to adapt to a broad range of attractant and repellent concentrations.     

Analysis of thyroglobulin iodination by tandem mass spectrometry using immonium ions of monoiodo- and diiodo-tyrosine

Peptides containing a monoiodo- or diiodo-tyrosine residue (monoiodo-Y, diiodo-Y) were found to generate abundant immonium ions following collision-induced dissociation at m/z 261.97 and 387.87 Da, respectively. These residue-specific marker ions are between about 140 mDa (monoiodo-Y) and 300 mDa (diiodo-Y) mass deficient relative to any other peptide fragment ions of unmodified peptides, qualifying them as highly specific marker ions for tyrosine iodination when analyzed by high resolution tandem mass spectrometry (MS/MS). Two new iodination sites (Y-364 and Y-2165) were pinpointed in bovine thyroglobulin by MS/MS using these iodotyrosine-specific marker ions and combined tryptic/chymotryptic digestion.     

Characterization of the two oligosaccharides present in the preferential hormonogenic domain of human thyroglobulin

The N-terminal fragment of human thyroglobulin (residues 1 to 171) contains the preferential hormonogenic site of the molecule and 2 potential sites of N-glycosylation (Asn57 and Asn91). This fragment was isolated from a human thyroglobulin purified from a single goiter. The tryptic peptides bearing the glycosylation sites were separated by Bio-Gel P-30 and HPLC columns. The oligosaccharides borne at each site were analyzed, after tritium labeling, by concanavalin A-Sepharose and HPLC. At both sites the structures observed are heterogenous, with a majority of biantennary complex type structures.     

Proton-magnetic-resonance studies on the interaction of rabbit skeletal-muscle troponin I with troponin C and actin.

1. The p.m.r. spectra of the larger CNBr-cleavage peptides of troponin I from rabbit fast-twitch skeletal muscle corresponded largely to those of fairly flexible solution structures. 2. On addition of troponin C to each of the CNBr-cleavage peptides in turn, perturbations of side chains were noted only for peptides CN5 (residues 1-21) and CN4 (residues 96-116). 3. In the presence of Ca2+, troponin C induced perturbations of the side chains of threonine-11, alanine, isoleucine and arginine residues of peptide CN5. 4. In the presence of Ca2+, troponin C induced perturbations of the side chains of phenylalanine, lysine and leucine residues of peptide CN4. 5. Irrespective of the presence or absence of Ca2+, specific interaction with actin was observed only with peptide CN4. In this case the side chains of arginine residues were perturbed. 6. It is concluded that actin interacts with the C-terminal region of peptide CN4, whereas troponin C interacts with the N-terminal region of peptide CN4 and with peptide CN5.     

Structural Proteins of Intracisternal A-Particles: Possible Repetitive Sequences

Peptide maps of tryptic digests of the structural proteins from inner shells of intracisternal A-particles have shown common peptides for all the proteins. The terminal amino group of the three different structural proteins was identified as arginine. The major protein revealed approximately half the number of peptides expected from the amino acid composition. Since evidence for a cross-link bond has not been found, the main structural protein may be a single polypeptide chain containing a total or partial duplication of sequence.     

The initiation of fetal hemoglobin biosynthesis.

Biosynthesis of the alpha and beta chains of rabbit and human adult hemoglobin is initiated with a methionyl residue, which is removed during elongation of the peptide chain. To study the initiation of biosynthesis of the delta chain of human fetal hemoglobin, fresh placental blood was used for labeling experiments with radioactive amino acids. Labeled nascent peptide chains were purified from the polysomal fraction of placental blood reticulocytes. The number of amino acid residues in nascent gamma chain at the time of removal of its N-terminal methionine was estimated to be 40--60 from the relative yields of labeled tryptic peptides.     

Proteomic analysis of mitochondria from the Bos taurus heart. II. Identification of mitochondrial soluble proteins

A fraction of the so-called mitochondrial soluble proteins was obtained after the destruction of purified mitochondria by sonication according to the previously found approach to the identification of protein subsets of the Bos taurus heart proteome. A tryptic destruction of these proteins was achieved. Approximately half of the tryptic hydrolysate was separated into two fractions of cysteine-containing and cysteine-free peptides by covalent chromatography on Thiopropyl Sepharose 4B. The cysteine-containing peptides were modified by iodoacetamide. The peptides were mass-spectrometrically identified in all the three fractions of tryptic hydrolysate, and the proteins were searched for in the amino acid sequence databases. There were 213 unique proteins reliably identified.     

Sites of covalent modification in Trg, a sensory transducer of Escherichia coli

The Trg protein mediates chemotactic response of Escherichia coli to the attractants ribose and galactose. Like other transducers, Trg is a transmembrane protein that undergoes post-translational covalent modification. The modifications are hydrolysis (deamidation) of certain glutamine side chains to create glutamate residues and methylation of specific glutamates to form carboxyl methyl esters. Analysis of radiolabeled, tryptic peptides by high performance liquid chromatography and gas-phase sequencing allowed direct identification of the modified residues of Trg. The protein has 5 methyl-accepting residues. Four, at positions 304, 310, 311, and 318, are contained in a 23-residue tryptic peptide ending in lysine. The fifth, at position 500, is within a 25-residue tryptic peptide ending in arginine. At two sites, 311 and 318, glutamines are deamidated to create methyl-accepting glutamates. There is not a required order of modification among the sites. However, there is a substantial preference for methylation on the arginine peptide and, among sites on the lysine peptide, for the middle pair. Comparison of sequences surrounding modified residues identified in this work for Trg and previously for Tsr and Tar suggests a consensus sequence for methyl-accepting sites of Ala/Ser-Xaa-Xaa-Glu-Glu*-Xaa-Ala/OH-Ala-OH/Ala, where OH signifies Ser or Thr and the asterick marks the site of modification.     

Trypsin cleaves exclusively C-terminal to arginine and lysine residues

Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity.     

Location of dehydroalanine residues in the amino acid sequence of bovine thyroglobulin. Identification of "donor" tyrosine sites for hormonogenesis in thyroglobulin

Thyroid hormonogenesis in thyroglobulin results in the conversion of an "acceptor" iodotyrosine to a hormone residue and a "donor" iodotyrosine to a dehydroalanine residue. Altogether five acceptor sites have been located as hormone residues in thyroglobulin of different animal species. To search for donor sites, we treated bovine thyroglobulin with 4-aminothiophenol to specifically modify dehydroalanine residues to S-(4-aminophenyl)cysteine (APC) residues, according to the principle of dehydroalanine determination developed by us (Kondo, T., Kondo, Y., and Ui, N. (1988) Mol. Cell. Endocr. 57, 101-106). After digesting thyroglobulin with lysyl endopeptidase, APC-containing peptides were separated from other peptides by trapping them on immobilized naphthylethylenediamine and from each other by size-exclusion and reverse-phase high performance liquid chromatography (HPLC). The HPLC patterns showed about 10 APC-containing peptides. Among them, four different peptides were purified by repeated reverse-phase HPLC. The results of partial sequencing of the four peptides by manual Edman degradation disclosed that Tyr5, Tyr926, Tyr1375, and Tyr986 or Tyr1008 are available for hormonogenesis as donor sites. These results strongly suggest that only specific tyrosine residues behave as donors.     

Carbonic anhydrase from spinach leaves. Isolation and some chemical properties.

Spinach carbonic anhydrase has been purified by modification and extension of a published method (Pocker, Y., and Ng. J. S. U. (1973) Biochemistry 12, 5127-5134), using (NH4)2SO4 precipitation and chromatography on DEAE-cellulose, agarose, and DEAE-Sephadex. The enzyme so obtained was homogeneous by criteria of both standard and sodium dodecyl sulfate polyacrylamide gel electrophoresis and of constant specific activity throughout the elution profile on DEAE-Sephadex chromatography. The enzyme has an apparent Mr of 212,000 by gel filtration on Sephadex G-200, a Mr of 26,000 by sodium dodecyl sulfate electrophoresis, and each of the subunits contains approximately 1 g atom of zinc. These data and the excellent correlation between the number of lysine and arginine residues per subunit, and the number of tryptic peptides obtained by peptide mapping, suggest that spinach carbonic anhydrase is an octamer consisting of identical or very similar subunits. Its amino acid composition is similar to parsley carbonic anhydrase; both contain large numbers of half-cystine residues relative to erythrocyte carbonic anhydrases. The spinach enzyme is devoid of disulfide bonds. The enzyme is stable around neutrality at -14 degrees, as a suspension in saturated (NH4)2SO4 solution.     

Small peptides controlling transcription in vitro are bound to chromatin DNA

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 micrograms/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.     

Radioimmunoassay for estimation of thyroglobulin in human serum.

A specific double antibody radioimmunoassay has been develop for the measurement of thyroglobulin in human serum. Human thyroglobulin was purified by combined DEAE-cellulose and affinity chromatography using Sepharose 4B-bound Concanavalin A. Sensitivity of test serum was 10 ng/ml. Thyroglobulin was not detectable in half of normal subjects, and half showed values between 10 and 180 ng/ml. In the patients with simple goiter and secondary hypothyroidism, serum thyroglobulin was usually in the normal range. In Hashimoto's thyroiditis, many sera having precipitating antibodies or high hemagglutination antibodies for thyroglobulin showed a high thyroglobulin concentration in serum probably due to a false positive reaction. In hyperthyroidism, an increased thyroglobulin level was observed in 64% of patients. However, there was no correlation between serum thyroglobulin and thyroxine levels in untreated hyperthyroidism. Serum thyroglobulin was increased significantly in some cases for several weeks after isotope therapy for the hyperthyroidism.