C-Terminal Sequence Analysis of Peptides Using Triphenylgermanyl Isothiocyanate

doi:10.1006/abio.2001.5505

Analytical Biochemistry

Volume 302, Issue 1, 1 March 2002, Pages 108-113

https://doi.org/10.1006/abio.2001.5505 Get rights and content

Abstract

The Schlack–Kumpf degradation, also called the isothiocyanate method, is thought to be a promising approach to chemical C-terminal sequencing of peptides and proteins. The derivatizing reagent is most crucial to this method. A new derivatizing reagent, triphenylgermanyl isothiocyanate (TPG-ITC), has been synthesized and applied to C-terminal peptide sequencing. The chemistry involves activation with acetic anhydride, derivatization with TPG-ITC, and cleavage of the derivatized C-terminal amino acid thiohydantoin with sodium hydroxide. A series of reaction conditions, including activation reagent volume, activation time, and derivatization temperature and time, have been investigated using a model peptide covalently attached to 1,4-phenylene diisothiocyanate (DITC)-glass beads. This procedure has been successfully used to sequence eight C-terminal residues of a model peptide at low nanomole levels. TPG-ITC is a white solid with relatively long shelf-life. According to our previous article (B. Mo, J. Li, and S. P. Liang, 1997, Anal. Biochem. 252, 169–176), TPG-ITC is a type II derivatizing reagent. Compared with acetyl isothiocyanate and trimethylsilyl isothiocyanate, TPG-ITC is much more stable and efficient for use in peptide C-terminal sequencing.

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Cited by (12)

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The determination of C-terminal peptide sequence is critical since the C-terminal peptide contains biologically relevant information and often undergoes post-translational processing. Another important application is in estimating purity of the biopharmaceuticals, especially for determining the presence of ragged processed ends and for N-terminally blocked polypeptides and proteins. In this paper, different isotope coding strategies in combination with reversed phase chromatography (RPC) coupled with electrospray ionization–mass spectrometry (ESI–MS) were evaluated to detect the C-terminal peptide from proteolytic digests. These were (i) O¹⁸ (ii) acylation and (iii) esterification based isotope coding strategies. Using reversed phase chromatography, the C-terminal peptide was resolved from other internal peptides. The isotope coding approaches specifically rendered a characteristic MS signature to the C-terminal peptide, thereby facilitating its detection. The unique MS signature, along with accurate mass data for the C-terminal peptide was found to be sufficient for its detection and identification. The advantages and limitations of the three approaches will be discussed.
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Fragmentation and “top–down” sequencing of intact proteins by mass spectrometry (MS) is most commonly performed by infusion of protein solutions into Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. However, the high cost of this instrumentation, coupled with the need to infuse “clean” solutions (lacking standard biological buffers), limits broad application of this technique. The current study describes an alternative approach to top–down sequencing using in-source fragmentation on quadrupole time-of-flight (Q-Tof) instrumentation coupled with reversed-phase liquid chromatography (LC). Application of this technique to purified recombinant samples yielded protein fragments during routine LC–MS analysis. The presence of multiple N- and C-terminal fragments allowed localization of structural modifications without proteolytic digestion. The method was extended to complex samples by using LC conditions that provided high-resolution protein separation. Utility of the method was illustrated by real-time monitoring of protein modifications occurring in reconstituted apoptosomes. These experiments illustrate that intact protein mass and limited sequence information can be obtained simultaneously on an LC timescale. This approach will allow a wide variety of laboratories to routinely apply top–down sequencing to problems in structural characterization, protein purification, and biomarker identification.
Mass spectrometric strategy for primary structure determination of N-terminally blocked peptides
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The mass spectrometric strategy including three steps is presented for primary structure determination of the N-terminally blocked peptides. First, the C-terminal sequencing is performed by using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry coupled with carboxypeptidase Y digestion. Then, the peptide is cleaved according to the obtained C-terminal sequence information and the resulting peptides are identified by mass spectrometry and Edman degradation after fractionation by reverse-phase chromatography. Finally, the N-terminal fragment is sequenced by tandem mass spectrometry. The strategy was successfully applied to the sequence determination of two novel N-terminally blocked peptides named EAFP1 and EAFP2.
Chapter 14 Chromatography of amino acids and peptides
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Regular ArticleC-Terminal Sequence Analysis of Peptides Using Triphenylgermanyl Isothiocyanate

Abstract

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Uber eine neue methode zur Ermittlung der konstitution von peptiden

Z. Physiol. Chem.

Regular Article
C-Terminal Sequence Analysis of Peptides Using Triphenylgermanyl Isothiocyanate