Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 3, 1995, pp. 167-168

REPORTE CORTO / SHORT REPORT

INFLUENCE OF BASIC RESIDUES ON THE C-TERMINAL REARRANGEMENT OF PEPTIDES IN GAS PHASE

Javier Gonzalez^1, Vladimir Besada^1, Hilda Garay^1, Osvaldo Reyes^1, Yanet Tambara^1, Toshifumi Takao^2, Yasutsugu Shimonishi^2 and Gabriel Padron^1.

^1Center for Genetic Engineering and Biotechnology. P.O. Box 6162, Havana, Cuba. ^2Institute for Protein Research. Osaka University, Osaka 565. Japan.

Code Number: BA95060
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We designed and synthesized a set of peptides in order to study some factors that could affect the appearance of this fragmentation such as the position of the basic residue within the sequence, the nature of the amino acids involved in the rearrangement and the way in wich the different basicity of two isobaric amino acids such as lysine and glutamine could be helpful to differentiate them taking into account the intensities ratio of bn-1/bn-1+18.

INTRODUCTION

The combination of mass spectrometry and soft ionization techniques have demonstrated its usefulness in protein sequencing due to its capability to handle complex mixture and sequence unknown and chemically modified peptides.

The interpretation of the Collision Activated Decomposition (CAD) spectra often tend to be complex, specially when intrinsic interactions regulate the fragmentation of peptides in gaseous phase. When basic amino acids are located within the peptide sequence or at the N-terminus, due to the C-terminal rearrangement signals appear in the CAD spectra originated by the interaction of the C-terminal hydroxyl group with the carbonyl group of the nearest amino acid (2). The peaks corresponding to this rearrangement has 18 mass units more than its correspondent bn serie and sometimes they are the most intense daughter ion in the spectrum such that specific sequences at the C-terminus could be misassigned.

We designed and synthesized a set of peptides in order to study some factors that could affect the appearance of this fragmentation such as the position of the basic residue within the sequence, the nature of the amino acids involved in the rearrangement and the way in wich the different basicity of two isobaric amino acids such as lysine and glutamine could be helpful to differentiate them taking into account the intensities ratio of bn-1/bn-1+18.

MATERIALS AND METHODS

Mass spectra were obtained with a JEOL JMS-HX11OHF two sector mass spectrometer operated with a JEOL JMA DA-5000 data system, ionization with a 4 KV Xenon beam produced positive ions that were analyzed at 10 kV accelerating voltage and resolution 1000. In the B/E linked-scan measurement Argon was used as a collision gas to decrease the intensity of the precursor ion to 50%.

All synthetic peptides were obtained using the multiple solid phase peptide synthesis. Dried and protected peptide-resin were treated with HF containing the appropriate scavenger mixture according to the Low-High procedure.

RESULTS AND DISCUSSIONS

The CAD spectra of peptides labeled with ^18O at their C-terminus shows signals with different isotopic ion distribution (3). The C-terminal ions show doublet signals, the N-terminal ions their natural isotopic distribution and the rearrangement ions a distribution different form the two pools previously mentioned allowing us an easier identification of this phenomenon and thus avoiding misassignments of the sequence.

Two set of pentapeptides were synthesized, one of them has a common amino acid at the C-terminus (Phe) and the unique change is at the penultimate amino acid, in the other set the penultimate amino acid was fixed (Ala) and the C-terminal was changed.

To facilitate the comparison between peptides we evaluated the intensity ratio b4+18/b4 because it shows the probability for both fragmentation to be formed. In the first set of peptides this parameter was similar for all peptides suggesting that the nature and the bulkiness of the side chain of the amino acid involved in the rearrangement do not have appreciable influences in this phenomenon with the exception of Pro as judge by the highest value of b4+18/b4. In the other set we observed light differences between peptides except when Lys and Arg are the amino acids lost during rearrangement wich suggest that the stability of the neutral molecules formed for this two amino acids are superior than the other.

We also synthesized five pentapeptides with the same amino acid composition, but the argine residue was located at different positions along the sequence and the results showed that this amino acid facilitated the rearrangement when it was located at position n-1.

Our results confirmed that this phenomenon is strongly influenced by the basicity of the amino acids within the sequence. In the CAD spectra of a peptide with the sequence KGIEF the ions of b4+18 and b4 has similar intensities, although for the peptide with Gln instead of Lys the rearrangement was almost insignificant. It was found that the appearance of this fragmentation allow to differentiate this two isobaric amino acids.

REFERENCES

1. GROSS, M. L. et al (1989). J. Am. Chem. Soc. 111:2835- 2842

2. GASKELL, S. J. et al . (1990). J. Am. Soc. Mass Spectrom. 1:249- 257

3. TAKAO, T. et al. (1991). Rapid Commun. Mass Spectrom. 5: 312- 318.

Copyright 1995 Sociedad Iberolatinamericana de Biotecnologia Aplicada a la Salud