Application of Mass Spectrometry to Protein Chemistry. I. Method for Amino-Terminal Sequence Analysis of Proteins.

Gray, William R.; Valle, U Del

doi:10.1021/bi00812a600

Cited by 26 publications

(11 citation statements)

References 12 publications

(11 reference statements)

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“…The drug and any of its metabolites retaining the labeled moiety showed an M, M + 3 doublet in the mass spectrum for the molecular ion and any fragment ions containing the labeled moiety. The concept of multiple deuterium labeling has been applied in sequence analysis of proteins by mass spectrometry (195,196).…”

Section: Methods-stablementioning

confidence: 99%

Studies with Deuterated Drugs

Blake

Crespi

Katz

1975

Journal of Pharmaceutical Sciences

113

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Section: Methods-stablementioning

confidence: 99%

Studies with Deuterated Drugs

Blake

Crespi

Katz

1975

Journal of Pharmaceutical Sciences

113

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“…By adopting a gas-liquid chromatography/ mass spectrometry (GLC-MS) micromethod recently developed by Rose et al (1983 b), we were able to determine the blocked amino-termini peptide sequences of both the VSV N and the Sendai virus NP proteins (Rose et al, 1983a). The basic technique (Gray & del Valle, 1970) involves acetylation, protease digestion and permethylation of the protein to produce volatile derivatives suitable for mass spectrometry. The GLC-MS techniques of Rose et al (1983a, b) should therefore be of general interest, as sequence information can be obtained directly from 2 to 5 nmol of proteins with blocked amino terminal.…”

Section: Introductionmentioning

confidence: 99%

Preparation and Analysis of the Nucleocapsid Proteins of Vesicular Stomatitis Virus and Sendai Virus, and Analysis of the Sendai Virus Leader-NP Gene Region

Blumberg

Giorgi

Rose

et al. 1984

Journal of General Virology

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SUMMARYA procedure is presented for isolating the nucleocapsid proteins, N and NP from vesicular stomatitis virus and Sendai virus respectively, in soluble form. These proteins were suitable for the determination of their blocked amino-terminal peptide sequences by gas-liquid chromatography/mass spectrometry at the low nanomole level. The N protein prepared by this procedure was previously shown to retain some of its expected biological activity. The sequence of 626 nucleotides from the 3' end of the Sendal virus genome, which includes the first one-third of the NP gene, was determined. Using this information, primer extension studies on intracellular Sendai virus mRNAs allowed the determination of the structure of the leader-NP intervening sequence and the 5' end of the NP mRNA. Comparison of the amino termini of the nucleocapsid proteins with their respective mRNA sequences revealed that these proteins are similarly processed in vivo. INTRODUCTIONThe non-segmented minus-strand (-) RNA genome of the rhabdovirus vesicular stomatitis virus (VSV), and that of the paramyxovirus Sendal virus, are always found tightly complexed in helical nucleocapsid structures with their respective viral nucleocapsid proteins, N and NP. The nucleocapsid structure protects the genomes from nuclease attack, and in conjunction with two less tightly associated viral proteins which constitute the viral RNA polymerase (NS and L of VSV: Emerson & Yu, 1975; P and L of Sendal: Stone et al., 1972;Marx et al., 1974; Lamb & Mahy, 1975), displays all the enzyme activities required for synthesis of the viral mRNAs (Banerjee et al., 1977;Kingsbury, 1974).Because the nucleocapsids are of central importance in both virus structure and replication, it would be of interest to study the properties of the purified nucleocapsid proteins themselves. However, previous attempts to isolate the N protein from nucleocapsids by treatment with dimethyl urea (S. Emerson, personal communication) or with guanidinium salts (G. Wertz, personal communication) have resulted in preparations with intractable physical properties. Both proteins can be removed from their nucleocapsids by treatment with SDS or phenol, but these reagents are likely to denature the protein. The viral nucleocapsids are also stable to highsalt conditions. Indeed, banding in CsC1 gradients at a buoyant density of 1.33 g/ml (2-5 M-CsC1) is diagnostic for VSV and Sendai virus nucleocapsids, for under these conditions all other cellular RNA-protein complexes disaggregate (Leppert et al., 1979). Thus, although these proteins are major components of the mature virion (Wagner et al., 1969; Mountcastle et al., 197t), they have resisted attempts at purification. In this paper, we describe a simple procedure for the isolation of N and NP proteins from intracellular nucleocapsids, based on CsCI density gradient centrifugation in the presence of guanidinium chloride.

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“…Electron impact spectra showed characteristic fragmentation along the peptide backbone around the carbonyl function of the (methylated) amide bond with retention of the charge on fragments containing the N-terminal residue. Examples of successful application of derivatization-GC/MS are the detection of N-and C-terminal sequences, particularly if the former are blocked and, thus, not amenable to Edman degradation (Gray & del Valle, 1970). New technologies in mass spectrometry that enlarged the range of peptide sequencing include Fast-atom bombard-FIGURE 5.…”

Section: A Mass Spectrometric Sequencing Of Peptidesmentioning

confidence: 99%

Contribution of mass spectrometry to contemporary immunology

Jong¹

1998

Mass Spectrom. Rev.

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Application of Mass Spectrometry to Protein Chemistry. I. Method for Amino-Terminal Sequence Analysis of Proteins.

Cited by 26 publications

References 12 publications

Studies with Deuterated Drugs

Studies with Deuterated Drugs

Preparation and Analysis of the Nucleocapsid Proteins of Vesicular Stomatitis Virus and Sendai Virus, and Analysis of the Sendai Virus Leader-NP Gene Region

Contribution of mass spectrometry to contemporary immunology

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