Sequencing of Peptide Mixtures by Edman Degradation and Field‐Desorption Mass Spectrometry

Shimonishi, Yasutsugu; Hong, Yeong‐Man; Kitagishi, Toyoko; Matsuo, T.; Matsuda, Hisashi; Katakuse, Itsuo

doi:10.1111/j.1432-1033.1980.tb07201.x

Cited by 54 publications

(13 citation statements)

References 22 publications

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“…In the past two decades, most interests in nanopores have focused on DNA sequencing. Only in recent years, nanopore technology has been extended to protein sequencing as nanopores could sequence full‐length proteins with high temporal and spatial resolution while traditional sequencing methods such as mass spectrometry and Edman degradation only rely on digestion of proteins into short peptides. Many experiments have shown that protein molecules can be electrophoretically driven through nanopores .…”

Section: Introductionmentioning

confidence: 99%

Discrimination of Protein Amino Acid or Its Protonated State at Single‐Residue Resolution by Graphene Nanopores

Zhang

et al. 2019

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cellular processes at a single-molecule level by taking advantage of the electrochemical nanoconfinement of a nanopore. Thus nanopore is a powerful sensor for investigation of fundamental physical, chemical or biological issues, such as molecule gating [5] and cation-induced current changing, [6] at single-molecule resolution [8] compared to other traditional technologies. To date, nanopores have been exploited for expanded applications, including detection of nanoparticles, [9][10][11] biopolymers, [1,12] proteins, [13][14][15] DNA-origami, [16] DNA-protein complexes, [17][18][19][20] and so on. One of the most potential applications of nanopore technology is nucleic acid sequencing, which is characterized as low cost and high throughput. Most recently, a lot of works have shown that each type of DNA nucleotide can induce correlated modulation of ionic current as they translocate through a nanopore. [12,[21][22][23] With the help of enzyme [24] or polymerase, [25] some biological nanopores [26,27] have shown the ability to determine the nucleotide sequence of a singlestranded DNA at single-base resolution.Different from DNA molecules, proteins consist of more than 20 amino acids (AAs) and are irregularly and slightly charged. Besides, proteins usually keep in folded state in natural environment. Thus, investigation of proteins is much more challenging than DNA. Unraveling the mechanisms of protein folding and unfolding and even realizing protein sequencing are essential for cellular behaviors and diseases. [28] Measurement of tunneling current [29,30] was reported to recognize single amino acids, however to make the tunneling device be a protein sequencing tool it should be coupled with a means that can thread the peptide chain through the gap in a controllable way, which could be possibly realized if the tunneling device is coupled to a nanopore. In the past two decades, most interests in nanopores have focused on DNA sequencing. Only in recent years, nanopore technology has been extended to protein sequencing as nanopores could sequence full-length proteins with high temporal and spatial resolution while traditional sequencing methods such as mass spectrometry [31] and Edman degradation [32] only rely on digestion of proteins into short peptides. Many experiments have shown that protein molecules can be electrophoretically driven through nanopores. [33,34] Some experiments showed that proteins can be unfolded using nanopores by adjusting pH values of the solution, [35] The function of a protein is determined by the composition of amino acids and is essential to proteomics. However, protein sequencing remains challenging due to the protein's irregular charge state and its high-order structure. Here, a proof of principle study on the capability of protein sequencing by graphene nanopores integrated with atomic force microscopy is performed using molecular dynamics simulations. It is found that nanopores can discriminate a protein sequence and even its protonation state at single-residue resolution. Both the pull...

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Section: Introductionmentioning

confidence: 99%

Discrimination of Protein Amino Acid or Its Protonated State at Single‐Residue Resolution by Graphene Nanopores

Zhang

et al. 2019

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“…The phenylthiohydantoin (Pth) derivatives of amino acids released were analyzed by HPLC and Pth-Lys and Pth-Asp were identified in the first and second steps of degradation, respectively. The peptide fragments were submitted to FAB mass spectrometry [4,23], giving signals at mjz = 1194.8 and 1079.7 after the first and second Edman degradations, respectively (data not shown). The mass differences between 1322.9 and 1194.8 and between 1194.8 and 1079.7 in each step of the degradation were 128.1 (Lys) and 115.1 (Asp), respectively.…”

Section: Other Methodsmentioning

confidence: 99%

Facile identification of protein sequences by mass spectrometry. B subunit of Vibrio cholerae classical biotype Inaba 569 B toxin

1985

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A mass spectrometric method was applied to the B subunit of Vibrio cholerae classical biotype Inaba 569B toxin to determine its amino acid sequence and to confirm the differences in the amino acid sequences predicted from the nucleotide sequences of the genes of El Tor biotype strains 62746 and 2125 toxins. In this method, the Staphylococcus aureus protease V8 digest of the CNBr-treated B subunit of the classical biotype toxin was examined directly by fast-atom-bombardment mass spectrometry without separation of individual peptides. The values of molecular ion signals observed in the mass spectra were compared with the amino acid sequences of the classical biotype and El Tor biotype toxins. All the observed mass values coincided with those calculated from the published sequences of the B subunit except those of the sequences at positions 12 -29 and 69 -79. Peptides with these sequences were isolated by high-performance liquid chromatography and analyzed by Edman degradation or by combination of mass spectrometry and enzymatic degradation. The results revealed that the amino acid residues at positions 22 and 70 were Asp instead of Asn in the published sequences of classical biotype toxin. It was also found that Asn at position 44 was partially deaminated to Asp. The amino acid sequence of the classical biotype toxin was found to be different only at positions 18 (His + Tyr), 47 (Thr + Ile) and 54 (Gly + Ser) from that of El Tor biotype toxins.In the last twenty years, mass spectrometric techniques have been used widely for determination of the amino acid sequences of proteins (for reviews see [l]). In particular, use of soft ionization mass spectrometric techniques, such as fielddesorption mass spectrometry [2] and fast-atom-bombardment (FAB) mass spectrometry [3] allows measurements of precise mass values of molecular and fragment ions of large underivatized peptides, resulting in facile determination of amino acid sequences. Recently, we developed a method for determining the amino acid sequences of proteins by a combination of soft ionization mass spectrometry and Edman degradation [4 -61 or carboxypeptidase digestion [7 -91 that does not involve separation of the peptides produced by digestion of proteins. Similar methods have been reported by others [lo, 111. As we have reported [6, 121, with our method almost all the molecular ion signals of constituent peptides in complex peptide mixtures produced by proteolytic digestion of proteins can be observed in the mass spectra. In mass measurements of several kinds of lysozymes the mass values of these signals coincided very well with those calculated from the amino acid sequences [ 121. Biemann [ 131 examined a chymotryptic digest of a tryptic peptide of neocarzinostatin by FAB mass spectrometry and, finding a difference between the observed mass values of the chymotryptic digest and those predicted from the published amino acid sequence of neocarzinostatin [14], he proposed a new amino acid sequence on the basis of mass measurements. Thus, direct measurement of ...

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“…Although mass spectral infonnation on peptides of Mr > 1000 had previously been obtained by their ionization with field desorption (FD) (Shimonishi et al, 1980) or plasma desorption (PDMS or 252Cf-MS) (Hakansson et al, 1982), FAB turned out to be a much simpler, more reliable, and more widely accessible technique for ionizing large peptides and soon even small proteins. Fortunately, the sensitivity of FAB-MS is high enough to be practically useful: 0.01-2 nmole (with increasing molecular size) is required for a measurement.…”

Section: Mass Spectrometric Methods For Determination Of the Structurmentioning

confidence: 99%

Protein Design and the Development of New Therapeutics and Vaccines

Hook¹,

Poste²,

Smith³

1990

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Sequencing of Peptide Mixtures by Edman Degradation and Field‐Desorption Mass Spectrometry

Cited by 54 publications

References 22 publications

Discrimination of Protein Amino Acid or Its Protonated State at Single‐Residue Resolution by Graphene Nanopores

Discrimination of Protein Amino Acid or Its Protonated State at Single‐Residue Resolution by Graphene Nanopores

Facile identification of protein sequences by mass spectrometry. B subunit of Vibrio cholerae classical biotype Inaba 569 B toxin

Protein Design and the Development of New Therapeutics and Vaccines

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