Protein fingerprint by fast atom bombardment mass spectrometry: Characterization of normal and variant human haemoglobins

Pucci, Piero; Carestia, Clementina; G, Fioretti; Mastrobuoni, A.M.; Pagano, L

doi:10.1016/0006-291x(85)90385-7

Cited by 64 publications

(28 citation statements)

References 14 publications

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“…Edman degradation steps were performed on HPLC-purified peptides, and were followed by the mass spectrometric analysis of the truncated peptides, in order to confirm the assignments, as already described (27).…”

Section: Methodsmentioning

confidence: 99%

Identification of Hormonogenic Tyrosines in Fragment 1218-1591 of Bovine Thyroglobulin by Mass Spectrometry

Gentile

Ferranti²,

Mamone³

et al. 1997

Journal of Biological Chemistry

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A fragment of bovine thyroglobulin encompassing residues 1218 -1591 was prepared by limited proteolysis with thermolysin and continuous-elution polyacrylamide gel electrophoresis in SDS. The reduced and carboxymethylated peptide was digested with endoproteinase Asp-N and fractionated by reverse-phase high performance liquid chromatography. The fractions were analyzed by electrospray and fast atom bombardment mass spectrometry in combination with Edman degradation. The post-translational modifications of all seven tyrosyl residues of the fragment were characterized at an unprecedented level of definition. The analysis revealed the formation of: 1) monoiodotyrosine from tyrosine 1234; 2) monoiodotyrosine, diiodotyrosine, triiodothyronine (T 3 ), and tetraiodothyronine (thyroxine, T 4 ) from tyrosine 1291; and 3) monoiodotyrosine, diiodotyrosine, and dehydroalanine from tyrosine 1375. Iodothyronine formation from tyrosine 1291 accounted for 10% of total T 4 of thyroglobulin (0.30 mol of T 4 /mol of 660-kDa thyroglobulin), and 8% of total T 3 (0.08 mol of T 3 /mol of thyroglobulin). This is the first documentation of the hormonogenic nature of tyrosine 1291 of bovine thyroglobulin, as thyroxine formation at a corresponding site was so far reported only in rabbit, guinea pig, and turtle thyroglobulin. This is also the first direct identification of tyrosine 1375 of bovine thyroglobulin as a donor residue. It is suggested that tyrosyl residues 1291 and 1375 may support together the function of an independent hormonogenic domain in the mid-portion of the polypeptide chain of thyroglobulin.Thyroglobulin (Tg), 1 a homodimeric glycoprotein with a molecular mass of 660 kDa, is the site of the biosynthesis of 3,5,3Ј-triiodothyronine (T 3 ) and 3,5,3Ј,5Ј-tetraiodothyronine (thyroxine, T 4 ) (reviewed in Ref. 1). T 3 and T 4 are synthesized via the iodination and coupling of a small subset of tyrosyl residues within the polypeptide chains of Tg. The coupling reaction takes place by the transfer of an iodophenyl group from a donor 3-monoiodotyrosine or 3,5-diiodotyrosine to an acceptor 3,5-diiodotyrosine. This causes the formation of T 3 or T 4 , respectively, at the acceptor site and dehydroalanine at the donor site (2, 3). Both reactions are catalyzed by thyroid peroxidase. Different tyrosyl residues have different reactivities toward iodine, so that iodination proceeds in a sequential order, which is controlled by the native structure of Tg (4, 5). Early iodinated tyrosyl residues are preferentially involved in iodothyronine synthesis (6); the coupling of iodotyrosines, in turn, has stringent steric requirements (7). In fact, out of 72 tyrosyl residues per bovine Tg monomer, only 15 are iodinated and a maximum of 6 -8 of them undergo coupling to form T 3 and T 4 (8, 9). So far, four major hormonogenic tyrosines have been identified, by the isolation and sequencing of hormone-rich peptides from Tgs of various animal species and comparison of their sequences with the cDNA-deduced sequences of bovine (10) and human Tg (11). Ty...

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Section: Methodsmentioning

confidence: 99%

Identification of Hormonogenic Tyrosines in Fragment 1218-1591 of Bovine Thyroglobulin by Mass Spectrometry

Gentile

Ferranti²,

Mamone³

et al. 1997

Journal of Biological Chemistry

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“…The diversity of dissociation channels observed within the charge state range studied potentially provides the opportunity to localize residues associated with variants via a top-down tandem mass spectrometry approach. M ass spectrometry has been applied to the characterization of hemoglobin and its variants for the past quarter of a century, beginning with the use of field desorption [1] and fast atom bombardment [2] as means for ionizing peptides derived from digestion. Today, applications of mass spectrometry to hemoglobin analysis generally employ either electrospray (ES) or matrix-assisted laser desorption ionization (MALDI) as the ionization method [3][4][5][6][7][8][9][10][11][12][13][14][15].…”

mentioning

confidence: 99%

Ion Trap Collision-Induced Dissociation of Human Hemoglobin α-Chain Cations

Mekecha

Amunugama

McLuckey

2006

J. Am. Soc. Mass Spectrom.

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Multiply protonated human hemoglobin ␣-chain species, ranging from [M ϩ 4H] 4ϩ to [M ϩ 20H] 20ϩ , have been subjected to ion trap collisional activation. Cleavages at 88 of the 140 peptide bonds were indicated, summed over all charge states, although most product ion signals were concentrated in a significantly smaller number of channels. Consistent with previous whole protein ion dissociation studies conducted under similar conditions, the structural information inherent to a given precursor ion was highly sensitive to charge state. A strongly dominant cleavage at D 75 /M 76 , also noted previously in beam-type collisional activation studies, was observed for the [M ϩ 8H] 8ϩ to [M ϩ 11H] 11ϩ precursor ions. At lower charge states, C-terminal aspartic acid cleavages were also prominent but the most abundant products did not arise from the D 75 /M 76 channel. The [M ϩ 12H] 12ϩ -[M ϩ 16H] 16ϩ precursor ions generally yielded the greatest variety of amide bond cleavages. With the exception of the [M ϩ 4H] 4ϩ ion, all charge states showed cleavage at the L 113 /P 114 bond. This cleavage proved to be the most prominent dissociation for charge states [M ϩ 14H] 14ϩ and higher. The diversity of dissociation channels observed within the charge state range studied potentially provides the opportunity to localize residues associated with variants via a top-down tandem mass spectrometry approach. (J Am Soc Mass Spectrom 2006, 17, 923-931)

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“…The material (500 pg) was then digested with trypsin (Sigma) as described elsewhere [20,211. FAB mass spectra were obtained on a VG ZAB 2 SE double-focusing mass spectrometer, fitted with a VG cesium gun operating at 25 keV (2 PA).…”

Section: Ms Analysismentioning

confidence: 99%

“…Peptide recognition within the amino acid sequence was accomplished using the FAB-mapping procedure previously described [22]. A single Edman degradation step was performed on the whole peptide digests, followed by FAB-MS analysis on the truncated peptides in order to confirm the assignments, as already described in [20].…”

Section: Ms Analysismentioning

confidence: 99%

“…Three rabbits (Martelli S.p. A., Florence, Italy) wcre subcutaneously injected with 20,50 or 100 pg purified re- analysis were collected on day 85. To assess anti-(recombinant protein) immunizations, 100 p1 rabbit immune sera, along with preimmune controls, properly diluted in 0.4% KC1,0.4% KH2P04, 16% NaCl and 2.5% NaH2P04, pH 7.2 (buffer A) containing 3% bovine serum albumin, were incubated at room temperature for 2 -3 h in multiwell flat-bottom RIA (Costar) strips previously coated with 200 p1 recombinant protein (2.4 pg/ml) in buffer A.…”

Section: Elisa Immunizations and Riamentioning

confidence: 99%

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Characterization by mass spectrometry of a recombinant hepatitis δ virus antigen and its proteolytic products

Tecce¹,

Petracca²,

Giuliani³

et al. 1992

European Journal of Biochemistry

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The recombinant hepatitis 6 virus antigen was obtained as a chimaeric protein fused to the Cterminus of the phage MS2 RNA polymerase. Following induction of the temperature-sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34-kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast-atom-bombardment mass spectrometry. The most represented degradation product, i. e. the 29-kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of 6 antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis 6 antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35 -40 amino acids at the C-terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti-(hepatitis 6 virus) antisera.Hepatitis 6 virus (HDV) is a defective single-stranded RNA virus which can only infect patients with acute or chronic infection by hepatitis B virus [l, 21. The hepatitis 6 antigen is essentially the only specific protein to be expressed as a consequence of HDV infection and which is immunogenic in infected patients [3]. It turned out that this protein is encoded by open reading frame (ORF) 5 in the viral genome and is specifically recognized by immune sera [4]. The diagnosis of hepatitis 6 virus infection is based both on the detection of the viral antigen and on the presence of the relative antibodies [l, 21. The availability of reproducible and safe preparations of the antigen is then necessary for the assembling of a clinical immunoassay for the detection of this viral disease. Viral preparations extracted from serum or liver tissue Now. The novel nucleotide sequence data published here have been submitted to the EMBL sequence data bank and are available under accession number X63373. The novel amino acid sequence data published here have been submitted to the EMBL sequence data bank. might be infective and yield a limited amount of material. To overcome these limitations, and to obtain from an unlimited and reproducible source a recombinant protein capable of substituting extractive antigens, the cloning and expression of the DNA sequence of hepatitis 6 antigen was undertaken.The recombinant protein was obtained together with several degradation products, similar to extracted hepatitis 6 antige...

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Protein fingerprint by fast atom bombardment mass spectrometry: Characterization of normal and variant human haemoglobins

Cited by 64 publications

References 14 publications

Identification of Hormonogenic Tyrosines in Fragment 1218-1591 of Bovine Thyroglobulin by Mass Spectrometry

Identification of Hormonogenic Tyrosines in Fragment 1218-1591 of Bovine Thyroglobulin by Mass Spectrometry

Ion Trap Collision-Induced Dissociation of Human Hemoglobin α-Chain Cations

Characterization by mass spectrometry of a recombinant hepatitis δ virus antigen and its proteolytic products

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