Affiliations:
1Mass Spectrometry Laboratory. Proteomics Department.
Center for Genetic Engineering and Biotechnology. Avenida 31 e/ 158 y
190. Cubanacán. Playa. PO. Box 6162. Havana. Cuba.
2Laboratory of Protein Organic Chemistry. Institute
for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka
565-0871, Japan.
3Thermo Fisher Scientific K.K. 3-9 Moriya-cho.
Kanagawa-ku, Yokohama-shi, Kanagawa, 221-0022, Japan.
4Animal Biotechnology Department. Center for Genetic
Engineering and Biotechnology. Avenida 31 e/ 158 y 190. Cubanacán.
Playa. PO. Box 6162. Havana. Cuba.
5Laboratory of Protein Profiling and Functional
Proteomics, Institute for Protein Research, Osaka University, 3-2
Yamadaoka, Suita, Osaka 565-0871, Japan.
(*) Authors with equal contributions
(§) Corresponding authors :
Dr. Toshifumi Takao
Laboratory for Protein Profiling and Proteomics, Institute for Protein
Research, Osaka
University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Phone: +816-6879-4312
Email:tak@protein.osaka-u.ac.jp
Dr. Luis Javier González López
Mass Spectrometry Laboratory. Department of Proteomics.
Center for Genetic Engineering and Biotechnology. Avenida 31 e/ 158 y
190. Cubanacán. Playa. PO. Box 6162. Havana. Cuba.
Email:luis.javier@cigb.edu.cu
Abstract :
RATIONALE: The thiosuccinimide linker is widely used in the
synthesis of bioconjugates. However, it is susceptible to hydrolysis and
is transformed into its hydrolyzed and/or the isobaric thiazine forms,
the latter of which is a fairly common product in a conjugate that
contains a cysteinyl peptide. MALDI-MS and MS/MS are useful for
differentiating these isobaric species.
METHODS: Four cross-linked peptides with thiosuccinimide
linkers were synthesized. Analogs with the linker that were transformed
into thiazine and/or the hydrolyzed thiosuccinimide linkers were then
generated by incubating the samples at neutral or basic pH. All of the
cross-linked peptides were purified by rp-HPLC and differentiated by
MALDI-MS, -MS/MS and UVPD.
RESULTS: A cysteinyl peptide-containing conjugate, the
thiosuccinimide form, was largely transformed into the hydrolyzed or
thiazine forms after incubation at neutral or basic pH. MALDI-MS allowed
the three forms to be differentiated: the thiosuccinimide and its
hydrolysis product gave two constituent peptides after reductive
cleavage between the Cys and succinimide moieties; no fragment ions were
produced from the thiazine form. In addition, MALDI-MS/MS of the
thiosuccinimide form yielded two pairs of complementary fragment ions
via 1,4-elimination: Cys-SH and maleimide, and dehydro-alanine and
thiosuccinimide, which are different from those produced via reductive
cleavage in MALDI-MS. The thiazine form gave fragment ions resulting
from the cleavage of the newly formed amide bond in the linker that
arose from thiazine formation.
CONCLUSIONS: The thiosuccinimide (but not thiazine) form of the
cross-linked peptide yielded individual constituent peptides in
MALDI-MS; MALDI-MS/MS showing specific 1,4-elimination for the
thiosuccinimide form and cleavage at the newly formed peptide bond via
transcyclisation for the thiazine form.