Sequencing of peptides and proteins from the carboxy terminus

Boyd, Victoria L.; Bozzini, MeriLisa; Zon, Gerald; Noble, Richard L.; Mattaliano, Robert J.

doi:10.1016/0003-2697(92)90376-i

Cited by 82 publications

(61 citation statements)

References 20 publications

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“…The thiohydantoin then becomes S-alkylated, transforming it into a good leaving group. During the subsequent cleavage (with the thiocyanate ion), the truncated protein forms a new thiohydantoin, eliminating the need for reactivation of the C-terminal carboxyl function during successive C-terminal sequencing (Boyd et al, 1992). Application of this chemistry to several proteins showed that the repetitive yield of the sequence analysis drops notably when encountering an Asp, Glu, or His residue, and that the sequence analysis stops at the C-terminal residue preceding Ser, Thr, and Pro.…”

Section: Benefits Of the Improved Chemical Approachmentioning

confidence: 99%

“…Some report that the secondary amino group does not allow the formation of the required oxazolinone (Matsuo et al, 1966;Holcomb et al, 1968). Stark pointed out that cyclization of proline thiohydantoin would require quaternization of the nitrogen atom involved in the peptide bond (Stark, 1968;Boyd et al, 1992). Bailey and Shively (1990) proposed that the lack of reactivity might be due to the insufficient nucleophilic character of the amide nitrogen of proline to attack the linear isothiocyanate or that the side chain of proline causes steric hindrance preventing cyclization of the five-membered thiohydantoin ring.…”

Section: Thiohydantoin Formation Goes Via the Oxazolinon Or The Oxazmentioning

confidence: 99%

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An improved chemical approach toward the C‐terminal sequence analysis of proteins containing all natural amino acids

et al. 1998

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An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N-acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 'H-NMR. This chemical method allows, for the first time, complete derivatization of N-acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C-terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C-terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C-terminal amino acids including proline is a major step forward in the development of a general chemical C-terminal sequencing method that permits the C-terminal sequence analysis of proteins of any amino acid composition.

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Section: Benefits Of the Improved Chemical Approachmentioning

confidence: 99%

Section: Thiohydantoin Formation Goes Via the Oxazolinon Or The Oxazmentioning

confidence: 99%

An improved chemical approach toward the C‐terminal sequence analysis of proteins containing all natural amino acids

et al. 1998

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“…Although C-terminal sequencing methods by stepwise degradation were developed (Inglis, 1991 ;Boyd et al, 1992;Bailey et al, 1995), no method is comparable to the Edman degradation for the N-terminus with respect to sensitivity and reliability. The two described strategies offer new possibilities for peptides in the low picomole range to obtain unambiguous information of the C-terminal amino acids by simple and straightforward techniques.…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless this method is limited because of the different preferences of the carboxypeptidases for each amino acid and each individual peptide bond, the formation of internal cleavages of the peptide chain and release of shorter peptides (Hayashi et al, 1975 Breddam, 1986). C-terminal degradation by using the thiohydantoin chemistry (Inglis, 1991 ;Bailey et al, 1992Bailey et al, , 1995 or the alkylthiohydantoin chemistry (Boyd et al, 1992) and analysis of the released amino acid derivatives by reverse-phase high performance liquid chromatography (RP-HPLC), and further, the exposure of the polypeptide to perfluoric acid (Tsugita et al, 1992) or perfluoracyl anhydride (Takamoto et al, 1995) and analysis by mass spectrometry are the most promising chemical procedures. Nevertheless, these methods have proven difficult and are yet limited to the upper picomole-to-nanomole range.…”

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confidence: 99%

C‐Terminal Ladder Sequencing by an Approach Combining Chemical Degradation with Analysis by Matrix‐Assisted‐Laser‐Desorption Ionization Mass Spectrometry

Thiede

Salnikow²,

Wittmann‐Liebold

1997

European Journal of Biochemistry

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A chemical procedure for the degradation of peptides and analysis by matrix-assisted-laser-desorption ionization mass spectrometry (MALDI-MS) was used for C-terminal sequence determination. The method allowed us to determine up to eight amino acid residues in the lower picomole range by mass analysis without any repetitions of the degradation nor any extraction or purification of the truncated peptide chains. The C-terminal degradation of all 20 common amino acid residues was proved by applying this method. Extended C-terminal sequence information from enzymatic digests using carboxypeptidase P was obtained by combining the enzymatic with the chemical mass spectrometric approach. Furthermore the amino acids lysine and glutamine, with the same masses, were distinguishable due to the formation of acetyl-lysine in the chemical process.Keywords: matrix-assisted-laser-desorption ionization MS ; C-terminal sequencing ; ladder sequencing ; enzymatic degradation. C-terminal sequence information is of general interest for the investigation of N-terminal-blocked peptides and proteins, for recombinant protein characterization, for the confirmation of DNA sequence data and for assistance in the design of oligonucleotide probes for cloning experiments.Several methods for C-terminal sequence determination have been developed, but no method has yet been described which is satisfactorily comparable to the Edman degradation for N-terminal sequencing (Edman and Begg, 1967;Hewick et al., 1981) in the low picomole range. Enzymatic degradation by carboxypeptidase digestion is the most commonly used method for Cterminal sequencing, either by determining released amino acids or by identifying the truncated peptides by mass spectrometry e.g. by fast-atom bombardment (Bradley et al., 1982), plasma desorption (Klarskov et al., 1989), electrospray ionization (Smith and Duffin, 1993) or matrix-assisted-laser-desorption ionization (MALDI-MS). The application of MALDI-MS (Karas and Hillenkamp, 1988) is particularly suitable for C-terminal sequence analysis after carboxypeptidase treatment, because of the high sensitivity, the relatively high tolerance to contamination and the easily interpretable data of mixtures. This method was called C-terminal ladder sequencing and was applied either by time-dependent (Thiede et al., 1995; Woods et al., 1995) or by concentration-dependent digestion (Patterson et al., 1995) and was used, e.g. for the identification of proteins separated by twodimensional gel electrophoresis (Thiede et al., 1996). Nevertheless this method is limited because of the different preferences of the carboxypeptidases for each amino acid and each individual peptide bond, the formation of internal cleavages of the peptide chain and release of shorter peptides (Hayashi et al., 1975 Breddam, 1986). C-terminal degradation by using the thiohydantoin chemistry (Inglis, 1991 ;Bailey et al., 1992Bailey et al., , 1995 or the alkylthiohydantoin chemistry (Boyd et al., 1992) and analysis of the released amino acid derivatives by reverse-pha...

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“…Chemical methods for C-terminal sequencing have been developed, but no method for the C-terminal sequence analysis has yet been described which works satisfactorily in cases of all amino acids [9][10][11], comparable with Edman degradation for the N-terminal region [12]. *Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

MALDI‐MS for C‐terminal sequence determination of peptides and proteins degraded by carboxypeptidase Y and P

Thiede¹,

Wittmann‐Liebold²,

Bienert³

et al. 1995

FEBS Letters

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Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for C-terminal amino acid sequence determination of peptides and proteins. The usefulness of MALDI-MS was demonstrated by analyzing peptide mixtures (C-terminal peptide ladder) which were generated by enzymatic digestion of substance P, glucagon, angiotensinogen, insulin B chain and myoglobin with the exopeptidases carboxypeptidase Y and P. The results clearly show that up to 11 amino acid residues can be determined in the pmol range by analyzing the molecular masses of the truncated peptides. For proteins it is possible to investigate enzymatic or chemical digests in the same manner.

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Sequencing of peptides and proteins from the carboxy terminus

Cited by 82 publications

References 20 publications

An improved chemical approach toward the C‐terminal sequence analysis of proteins containing all natural amino acids

An improved chemical approach toward the C‐terminal sequence analysis of proteins containing all natural amino acids

C‐Terminal Ladder Sequencing by an Approach Combining Chemical Degradation with Analysis by Matrix‐Assisted‐Laser‐Desorption Ionization Mass Spectrometry

MALDI‐MS for C‐terminal sequence determination of peptides and proteins degraded by carboxypeptidase Y and P

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