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\begin{document}

\title{Differentiation of isobaric cross-linked peptides prepared via maleimide
chemistry by MALDI-MS and MS/MS.}



\author[1]{Toshifumi Takao}%
\author[2]{Luis Gonzalez}%
\author[2]{Satomy Pousa}%
\author[1]{Hironobu Hojo}%
\author[3]{Shio Watanabe}%
\author[3]{Daisuke Higo}%
\author[2]{Alina Rodriguez Mallon}%
\affil[1]{Osaka Daigaku Tanpakushitsu Kenkyujo}%
\affil[2]{Centro de Ingenieria Genetica y Biotecnologia}%
\affil[3]{Thermo Fisher Scientific Inc Mitato-ku Tokyo}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{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. \textbf{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.
\textbf{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. \textbf{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.%
\end{abstract}%



\sloppy


\textbf{Title:} Differentiation of isobaric cross-linked peptides
prepared via maleimide chemistry by MALDI-MS and MS/MS.

\textbf{Short Title:} Differentiation of thiazine and thiosuccinimide
linkers by MS.

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/654937/articles/660997-differentiation-of-isobaric-cross-linked-peptides-prepared-via-maleimide-chemistry-by-maldi-ms-and-ms-ms}

\textbf{Auhtors:} Luis Javier Gonz\selectlanguage{ngerman}ález\textsuperscript{1,*,§}, Satomy
Pousa\textsuperscript{1,*}, Hironobu Hojo\textsuperscript{2}, Shio
Watanabe\textsuperscript{3}, Daisuke Higo\textsuperscript{3}, Alina
Rodriguez Mallon\textsuperscript{4} and Toshifumi
Takao\textsuperscript{5,§}

\textbf{Affiliations:}

\textsuperscript{1}Mass Spectrometry Laboratory. Proteomics Department.
Center for Genetic Engineering and Biotechnology. Avenida 31 e/ 158 y
190. Cubanacán. Playa. PO. Box 6162. Havana. Cuba.

\textsuperscript{2}Laboratory of Protein Organic Chemistry. Institute
for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka
565-0871, Japan.

\textsuperscript{3}Thermo Fisher Scientific K.K. 3-9 Moriya-cho.
Kanagawa-ku, Yokohama-shi, Kanagawa, 221-0022, Japan.

\textsuperscript{4}Animal Biotechnology Department. Center for Genetic
Engineering and Biotechnology. Avenida 31 e/ 158 y 190. Cubanacán.
Playa. PO. Box 6162. Havana. Cuba.

\textsuperscript{5}Laboratory of Protein Profiling and Functional
Proteomics, Institute for Protein Research, Osaka University, 3-2
Yamadaoka, Suita, Osaka 565-0871, Japan.

(*) Authors with equal contributions

(\textsuperscript{§}) \textbf{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:\emph{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:\emph{luis.javier@cigb.edu.cu}

\textbf{Abstract} :

\textbf{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.

\textbf{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.

\textbf{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.

\textbf{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.

\textbf{Keywords:}

thiosuccinimide linker, thiazine linker, transcyclization, MALDI-MS.

\textbf{Introduction:}

The thiol-maleimide reaction \textsuperscript{1} also known as the
thiol-Michael addition \textsuperscript{2}, is included among the
chemical reactions classified as click-chemistry
\textsuperscript{2,3}and is one of the most widely used methods for
conjugation\textsuperscript{4,5} and the synthesis of antibody drug
conjugates (ADC) and conjugate vaccines \textsuperscript{4}.

As a result of this chemical reaction, the cytotoxic drug and the
antigen are linked to the carrier protein through a thioether bond,
generally between a cysteine residue, a five-membered succinimide ring
and an \emph{N} -acyl group that is linked to primary amino groups
(structure I, Figure 1). The resultant five membered thiosuccinimide
ring is considered to be unstable \emph{in vivo} due to the fact that a
retro-Michael addition can occur \textsuperscript{6,7} (Figure S1). In
serum, the presence of molecules with free thiols (e.g. free Cys,
reduced glutathione, albumin, etc.), through a thiol-exchange reaction,
promotes the release of the antigen or the cytotoxic drug linked to the
thiosuccinimide ring in the conjugate vaccine and ADC, respectively.
This reaction, which is undesirable for the development of a
biotechnology product, increases the cytotoxicity of the
ADC\textsuperscript{8}, and it could also lead to the partial or
complete loss of the biological activity of the conjugate vaccine. To
overcome this potential \emph{in vivo} instability of the resultant
five-membered thiosuccinimide ring, it can be stabilized through
hydrolysis\textsuperscript{9-13} or via Michael-transcyclization
reactions\textsuperscript{14,15}.

Hydrolysis of the five-membered thiosuccinimide could proceed through
two pathways (see pathways (a) and (b) in structure (I), Figure 1) that
would yield two thiosuccinamic acid isomers (structures (II) and (III),
Figure 1) \textsuperscript{10}. These isomers are stable \emph{in
vivo}and are not subject to the retro Michael addition. This hydrolysis
of the thiosuccinimide linker proceeds more rapidly at basic pH, and
also takes place during the sample preparation and proteolysis of
conjugates regardless of the position of the cysteine residue that is
involved in the thioether bond \textsuperscript{16-18}. The
thiosuccinamic acids, hydrolysis products derived from a
thiosuccinimide, can be readily identified by the MS-mode, based on the
fact that the molecular masses of these species differ by 18 Da.

In the case of the peptide with a free cysteine at the \emph{N}terminus,
the transcyclization predominantly takes place by a nucleophilic attack
of the amino terminal group of the cysteine residue to the carbonyl
group in the five-membered ring of the thiosuccinimide, which, through
an internal rearrangement, results in the formation of a more stable
six-membered thiazine ring and a new pseudopeptide bond as well
(structure (IV), in Figure 1) \textsuperscript{14}.

The conjugate peptides linked through thiazine or thiosuccinimide are
isobaric, and thus, cannot be differentiated based on their molecular
masses. However, the thiazine form can be identified by CID or HCD
fragmentation\textsuperscript{15}. This is because the thiazine form can
undergo cleavage at new pseudo-peptide bond that was formed through the
transcyclization process\textsuperscript{14} (see structure (IV), in
Figure 1).

The findings reported in this study show that C-S linkage between the
cysteine sulfur atom and the succinimide at the conjugation site is
amenable to cleavage by MALDI-MS, MALDI-MS/MS or UVPD/HCD-MS/MS, which
yields two component peptides, namely, a free-thiol-containing peptide
and its counterpart linked to the original \emph{N} -propionyl maleimide
and \emph{N} -propionyl succinimide groups. The thiazine form, however,
is stable and no fragment ions derived from it are produced in MALDI-MS,
but fragment ions for the newly formed peptide bond are observed in
MALDI-MS/MS.

\textbf{Materials and Methods:}

\textbf{Peptide Synthesis}

Starting from the Fmoc-Lys(Boc)-OCH\textsubscript{2}-Wang resin (0.175
g, 0.1 mmol, 0.57 mmol/g), the peptide was elongated by CEM Liberty Blue
using a 0.1 mmol scale protocol to give an Fmoc-AAGGGAAAAK(Boc)-resin.
The resulting resin was divided into two portions and one was used for
the further synthesis to obtain the Fmoc-AAAGGGAAAAK-resin (P1 peptide).
For the other, Fmoc-C(Trt)-OH (0.25 mmol act by HBTU 0.25 mmol, DIEA 0.5
mmol) was reacted for 30 min at 50 ºC to give the
Fmoc-C(Trt)AAGGGAAAAK(Boc)-resin (P2 peptide).

Starting from the Fmoc-Arg(Pdf)-OCH\textsubscript{2}-PEG-resin (0.23 g,
0.05 mmol, 0.22 mmol/g), the peptide chain was elongated by CEM to
obtain
Ac-GAN(Trt)APK(Boc)E(OBut)PQ(Trt)R(Pbf)-OCH\textsubscript{2}-PEG-resin
(P4 peptide). Starting from the Fmoc-Lys(Boc)-OCH\textsubscript{2}-Wang
resin (0.0875 g, 0.05 mmol, 0.57 mmol/g), the peptide chain was
elongated by CEM to obtain
NH\textsubscript{2}-N(Trt)C(Trt)AGH(Trt)K(Boc)-OCH\textsubscript{2}-Wang-resin
(P3 peptide).

The deprotected P1 peptide (AAAGGGAAAAK) anchored to the dried resin was
stirred with a 20 \% piperidine solution at room temperature for 30
minutes, washed with DMF and dried under a vacuum. The peptide-resin was
treated with an excess of the non-cleavable heterobifunctional protein
cross-linker BMPS ((N-\selectlanguage{greek}β-\selectlanguage{english}maleimidopropyloxy)succinimide ester) from MERCK
dissolved in DMF spiked with traces of trimethylamine and the reaction
proceeded during a period of 30 minutes. The resin was washed with DMF
and dried under a vacuum. In the case of P4, it was dissolved in 100 mM
Tris/HCl buffer, pH=6.8 and treated with an excess of a solution of BMPS
in DMF. The reaction proceeded for 30 minutes and the P4 peptide
modified with a maleimide group at the \selectlanguage{greek}ε-\selectlanguage{english}amino group of Lysine residue
was purified by rp-HPLC.

All peptides anchored to the resin were deprotected and cleaved from the
resin by treatment with a mixture containing TFA:H\textsubscript{2}O:TMS
in a ratio of 95:2.5:2.5 (v/v). The peptides were precipitated with
diethyl ether, the excess diethyl ether was removed by centrifugation
and the peptide pellet was dried under a stream of nitrogen. These
peptides were dissolved in H\textsubscript{2}O/TFA (0.1 \% v/v) and
purified by rp-HPLC.

As a result, four peptides were synthesized: P1 (*AAAGGGAAAAK), P2
(CAAGGGAAAAK), P3 (NCAGHK), and P4 (Ac-GANAPK*EPQR). Asterisks in the
sequences of P1 and P4 peptides mean that their primary amino groups
were modified with an \emph{N} -propionyl maleimide group. Ac- in the P4
peptide sequence indicates that an acetyl group is located at
the\emph{N} -terminal end.

Cross-linked peptides were synthesized by the maleimide-thiol
chemistry\textsuperscript{1,2}. Approximately equal amounts of the free
Cys containing peptides (P2 and P3) and the maleimide \emph{N}
-propionyl maleimide-containing peptides (P1 and P4) were dissolved at
equal concentrations in 100 mM Tris/HCl, pH=6.8 and mixed in the correct
combinations to synthesize four cross-linked peptides with the
thiosuccinimide linker. These compounds are referred to hereafter as
(P1-P2), (P1-P3), (P3-P4), and (P2-P4). The reactions proceeded during
20 minutes at room temperature with stirring. The final products of the
reactions were analyzed by rp-HPLC and the completion was confirmed by
the fact that one of the starting peptides had been completely consumed.
The new generated peak fractions that potentially contained the desired
product were collected and directly analyzed by MALDI-MS.

\textbf{Preparation of cross-linked peptides with the linker transformed
to thiazine and hydrolyzed thiosuccinimide variants.}

Cross-linked peptides were dried in a speed-vac, dissolved in 100 mM
Tris-HCl buffer, pH=8.0, and incubated at 37\textsuperscript{o}C for 6
hours to transform them into a thiazine and/or hydrolyzed
thiosuccinimide forms that correspond to P3-P4 in Figure S2, P1-P3 in
Figure S3, P2-P4 in Figure S4, and P1-P2 in Figure 2. All of the
transformed forms were purified by rp-HPLC, collected, and analyzed by
MALDI-MS, -MS/MS and LC-MS/MS embedded with UVPD (see below).

\textbf{RP-HPLC analysis}

The peptides were purified with an HP1100 HPLC system (Agilent
Technologies, Inc) on a column (ZORBAX Eclipse Plus C18 RRHD 2.1x50 mm,
1.8 \selectlanguage{greek}μ\selectlanguage{english}m, Agilent Technologies, Inc) that had been equilibrated in 98 \%
of solution A (0.1 \% v/v TFA in ultrapure water). The concentration of
solution B (TFA, 0.1 \% v/v in pure ACN) in the mobile phase was
increased to 55 \% in 15 minutes and reached 80 \% of solution B in 0.1
minutes. The column was washed for an additional 0.5 min in 80 \% of
solution B and was then equilibrated under the initial conditions for a
new separation. The flow rate was 0.35 mL/min and the absorbance was
monitored at 210 nm during the chromatographic separation. The
temperature of the column was maintained at during all chromatographic
separations.

\textbf{MALDI-MS analysis}

MALDI-TOF MS was carried out with a 4800 MALDI-TOF/TOF mass spectrometer
from Applied Biosystems (Framingham, MA, USA). All mass spectra were
obtained by averaging 2500 laser shots from each sample well in the
positive-ion mode. The instrument was controlled using the 4000 series
Explorer software (version 3.6; Applied Biosystems). Data were processed
by using Data Explorer software (version 4.8; Applied Biosystems). The
calibration of the instrument in the MS and MS/MS modes were carried out
by using the ions derived from a mixture of several synthetic peptides
(Angiotensin I, 1296.685; Dynorphin A, 1603.991; ACTH (1-24), 2932.588;
beta-endorphin, 3463.830), and several product ions derived from
angiotensin I, respectively. Ordinary MS/MS spectra were obtained with a
fully automatic workflow, in which the metastable suppressor and CID gas
(air) were both activated.

Peptides dissolved in water/acetonitrile/TFA solutions (0.5 \selectlanguage{greek}μ\selectlanguage{english}L) were
mixed with the (0.5 \selectlanguage{greek}μ\selectlanguage{english}L) matrix solution, (the supernatant of a 50 \%
acetonitrile solution saturated with \selectlanguage{greek}α--\selectlanguage{english}cyano-4--hydroxycinnamic acid),
and then air dried on the flat surface of a stainless steel plate. The
ions were generated by irradiating the sample area with the output of an
Nd/YAG laser at a wavelength of 355 nm, and the resulting generated ions
were accelerated at a 20 kV potential in the ion source.

\textbf{LC-MS/MS with UVPD fragmentation}

Peptide fragmentation by UVPD was carried out in an Orbitrap Eclipse
Tribrid mass spectrometer (Thermofisher Scientific, Waltham, MA). Data
were acquired by the DDA method and Orbitrap detection. MS1 was
collected in the mass range from m/z 375 to \emph{m/z} 1500 at a 120,000
resolution with an AGC target of 1 x 10\textsuperscript{6} and a maximum
injection time of 100 ms. Followed by MS1, MS2 was collected at a 30,000
resolution with an AGC target of 1 x 10\textsuperscript{5} and a maximum
injection time of 55ms. Each precursor was isolated with a 2.0 Th
window. The peptides were fragmented by laser irradiation in the central
part of the quadrupolar dual linear ion trap, and the fragment ions were
transferred to the Orbitrap mass analyzer for measurement of their
molecular masses. Peptides were photoactivated during 500 ms with the
fifth harmonic Q-switched 213 nm Nd:YAG laser (CryLaS, GmbH). Peptides
were applied to the Vanquish Neo UHPLC system (Thermo Fisher Scientific,
Waltham, MA), and separated on a 75um ID x 125mm capillary column
(Nikkyo Technos, Tokyo, Japan) with a linear gradient from 0\% to 45\%
of solvent A/B (A : water with 0.1\% formic acid; B : 80\% acetonitrile
with 0.1\% formic acid (Thermo Fisher Scientific, Waltham, MA)) for 10
minutes (synthetic peptides) or 60 minutes (conjugates) at a flow rate
of 300 nL/min and sprayed into the mass spectrometer with a 2000 V spray
voltage as the ESI.

\textbf{Results and Discussion:}

MALDI-MS analyses of cross-linked peptides with a thiosuccinimide linker
and their hydrolyzed variants.

Considering the integrity of structural variants (hydrolyzed and
thiazine forms\textsuperscript{17,18}) of the linker moiety, which are
relevant to the conjugation modes, four types of cross-linked peptides
were prepared (Table 1). P3-P4 and P1-P3 gave only the hydrolyzed forms
upon incubation under alkaline conditions (see Materials and Methods).
P1-P2 and P2-P4 both gave the hydrolyzed and thiazine forms, the latter
of which was the product of transcyclization between the\emph{N}
-terminal Cys of P2 and succinimide (see Figure 1).

The rp-HPLC profile of the P1-P2 cross-linked peptide showed a major
fraction at 4.86 min (see the lower chromatogram in Figure 2). MALDI-MS
analysis of this fraction (Figure 3a) confirmed the identity of this
molecule. Intense signals at \emph{m/z} 1812.88 and \emph{m/z} 906.96
were assigned to (M+H)\textsuperscript{+} and
(M+2H)\textsuperscript{2+}ions of this cross-linked peptide with the
thiosuccinimide linker, respectively (Table 1).

The signals at \emph{m/z} 847.43 and \emph{m/z} 968.50 had an
appreciable intensity and were assigned to the P2 peptide with a free
thiol and the P1 peptide linked to an \emph{N} -propionyl succinimide,
respectively (Figure 3a, structure (II) in 3d), which were confirmed by
MS/MS analysis (Figures S5 and S6a) and were most likely produced via
reductive cleavage during the MALDI procedure. In the MALDI-MS analysis
of the crosslinked peptides prepared with other crosslinker reagents
based on similar maleimide-thiol chemistry, signals resulting from
cleavage of the thioether bond were also
observed\textsuperscript{19,20}.

Note that an expanded region of this MALDI-MS spectrum (see inset Figure
3a) also showed the presence of an ion at \emph{m/z} 966.49, which
corresponds to the P1 peptide, but with an \emph{N} -propionyl maleimide
group (structure (I) in Figure 3d). This fragment ion could be generated
via a 1,4-elimination reaction at the thioether bond as depicted in
Figure 4a, which was also observed by MS/MS analysis (Figure S6b).

The P1-P2 peptide but with a hydrolyzed linker, referred to as
(P1-P2)\textsubscript{H} (rt. 4.70 min in the upper chromatogram, Figure
2), was prepared by incubating the original P1-P2 peptide (rt. 4.86 min
in Figure 2, lower chromatogram) at pH=8.0 for 6 hours at
37\textsuperscript{o}C. Its molecular mass was observed at
\emph{m/z}1830.64 (Figure 3b), the mass of which was larger by 18 Da
than the original one (Figure 3a), suggesting that either of the two
imide bonds in the thiosuccinimide had been hydrolyzed by the hydrolytic
process (see structure (I) in Figure 1). It is noteworthy that the
signals at\emph{m/z} 847.32, 984.36 and 986.38 in Figure 3b could be
assigned to the P2 peptide with a thiol and the P1 peptide with the
counterpart but with the hydrolyzed linker (structure (III) and (IV) in
Figure 3d), which were likely produced via 1,4-elimination or reductive
cleavage, respectively, as observed for (P1-P2).

The above results clearly suggest that the linkage between the
succinimide ring and Cys could be cleavable upon MALDI, the fact of
which is also supported by the results obtained for the other
cross-linked peptides, three of them with the thiosuccinimide linker
(P1-P3), (P2-P4) and (P3-P4), and their corresponding analogs with a
hydrolyzed thiosuccinimide linker, (P1-P3)\textsubscript{H},
(P2-P4)\textsubscript{H} and (P3-P4)\textsubscript{H} (see Figures
S7-S9).

Considering the strong absorption of laser energy by hot matrices such
as \selectlanguage{greek}α-\selectlanguage{english}CHCA in MALDI and the subsequent transfer of energy to the analyte,
it can be assumed that the excess energy stored in the analyte ions led
to the spontaneous dissociation of the thioether bond, which is a
relatively weak chemical bond. This result is consistent with the MALDI
ionization method and suggests that thioether-based linkers are
cleavable when the MALDI ionization method is used in such an analysis,
and consequently provides useful information concerning the components
of the cross-linked peptide.

Differentiation by MALDI-MS and -MS/MS analysis of cross-linked peptides
with thiosuccinimide and thiazine linkers.

The MALDI-MS spectra of cross-linked peptides (P1-P2) and (P2-P4) with a
thiosuccinimide linker were compared with their corresponding isomers
with a thiazine linker. Both of the cross-linked peptides with the
thiosuccinimide linker gave a signal at \emph{m/z} 847 which corresponds
to the thiol-containing peptide (P2-SH). Their counterparts, linked to
the \emph{N} -propionyl maleimide or \emph{N} -propionyl succinimide,
were observed at \emph{m/z} 966.49 and 968.50 (inset of Figure 3a), and
at\emph{m/z} 1260.32 and 1262.33 (inset of Figure 5a), which were
assigned to P1-(I) and P1-(II) in Figure 3d, and P4-(I) and P4-(II) in
Figure 5c, respectively. As in the case of P1* in the (P1-P2)
cross-linked peptide (see Figure 3a), the P4 peptide was observed as
\emph{N} -propionyl maleimide or \emph{N} -propionyl succinimide forms
(Figure 5c), which arose from 1,4-elimination or reductive cleavage,
respectively, at the C-S linkage between the Cys residue and the
succinimide during the MALDI process.

The cross-linked peptides, P1-P2 and P2-P4, gave significant amounts of
the isobaric thiazine forms under coupling conditions (Figure 2 and
Figure S4) and are denoted as (P1-P2)\textsubscript{T} and
(P2-P4)\textsubscript{T}. As described above, this transformation from
the thiosuccinimide linker predominantly occurs in a spontaneous manner
for the case where a conjugated peptide contains an \emph{N} -terminal
Cys. The molecular mass of the thiazine form was the same as that of the
original thiosuccinimide form, but none of the fragment ions
characteristic of the peptide with a thiosuccinimide linker were
observed (Figures 3c and 5b). This result suggests that the thiazine
linker is stable and resistant to cleavage during MALDI while the
thiosuccinimide linker or its hydrolysis form is amenable to cleavage at
the C-S linkage of the linker (Figures 3a and 5a).

These results suggest that the thiosuccinimide forms, including those
that had been hydrolyzed, can be unambiguously identified by observing
the peptides constituting the conjugate simultaneously in MALDI-MS (see
Figures 3a, 3b, 5a, and S7-S9). In contrast, the thiazine form does not
yield these peptide ions (Figures 3c and 5b). This suggests that the
thioether bond, embedded in the six-membered ring of thiazine, is not
susceptible to such cleavage of the conjugate that yields the two
constituent peptides.

MALDI-MS/MS was then examined in an attempt to distinguish these
isobaric peptides. The MALDI-MS/MS spectra of (P1-P2) with
thiosuccinimide and (P1-P2)\textsubscript{T} with the thiazine linker
were nearly identical, with respect to the \emph{m/z} values of the
backbone fragment ions for the P1 and P2 peptides (Figures 6a and 6d,
Figures S10 and S11). However, there was a marked difference in the
expanded region (\emph{m/z} 792-1013, Figures 6b and 6c). The
thiosuccinimide form (P1-P2) yielded two pairs of complementary fragment
ions that were produced via a 1,4-elimination: Cys-SH and maleimide
(Figure 4a), and dehydro-alanine and thiosuccinimide (Figure 4b), which
was distinctly different from those produced via reductive cleavage in
MALDI-MS (see Figure 3a). The other cross-linked peptides, (P1-P3) and
(P3-P4), with the thiosuccinimide linker produced fragment ions that
were similar to those observed for (P1-P2) (see Figures S12 and S13).

In the case of (P1-P2)\textsubscript{T}, the thiazine form, which is
formed primarily when a cysteinyl peptide is cross-linked to another
peptide containing Lys by BMPS (upper chromatogram in Figure 2,
retention time 5.10 min), no ions were observed when the above C-S bond
was cleaved. Instead, a pair of ions at \emph{m/z} 886.36 (P1+71) and
its counterpart of ion at \emph{m/z} 927.30 (P2+L-71, where L denotes
`linker'), highlighted in red, were newly observed (Figures 6c and 6d).
These ions, specifically observed for (P1-P2)\textsubscript{T}, were
those produced when cleaved at the new pseudo-peptide bond generated in
the thiazine-containing linker (Figure 1, structure (IV)). Similar
results were also obtained for (P2-P4)\textsubscript{T} (see Figures S14
and S15).

The above results demonstrate that MALDI-MS/MS allowed both isobaric
cross-linked peptides with thiosuccinimide and thiazine linkers to be
clearly differentiated, based on the fragment ions produced via a
1,4-elimination at the thiosuccinimide/Cys residue and the fragmentation
of the newly formed pseudopeptide bond in a thiazine linker,
respectively, the latter of which has also been observed by LC-MS/MS
using HCD as a fragmentation method \textsuperscript{15}.

UVPD fragmentation of cross-linked peptides with thiosuccinimide and
thiazine linkers

Ultraviolet photodissociation (UVPD) has been applied for the
identification of disulfide heterogeneity and non-native crosslinks such
as trisulfide and thioether bonds which are generated during the
production of and storage of monoclonal antibodies \textsuperscript{21}.
UVPD at 213 nm can be used to selectively dissociate these bonds to
produce characteristic fragmentation patterns which are derived from the
homolytic cleavage of S-S and C-S linkages \textsuperscript{22}. Based
on these previous observations, we examined the issue of whether or not
conjugates containing thiosuccinimide or thiazine linkers could be
distinguished by the UVPD method incorporated in LC/ESI-MS/MS (see
Experimental section). For P2-P4, product ions at m/z 813.42, 847.41 and
1260.60 in Figure S16a, observed for the thiosuccinimide form, were
similar to those observed in MALDI-MS/MS (Figure S14), which most likely
had arisen from a 1, 4-elimination (Figure 4). Note that the observed
product ions were not those derived from homolytic cleavage at the C-S
linkage of the thiosuccinimide, since they were smaller by 1 Da than
those from the homolytic cleavage. These ions were sufficient for
identifying the thiosuccinimide form, whereas, UVPD-MS/MS of the
thiazine form did not give any product ions derived from the cleavage at
the pseudopeptide bond which was newly formed in the thiazine form
(Figure S16b). Considering the conditions of the UVPD used in this
study, i.e., photoactivation time (500 ms) and the method of ion
trapping in the device, it is likely that the precursor ions primarily
underwent spontaneous fragmentation by 1, 4- elimination rather than
dissociation by UV-laser irradiation.



\textbf{Conclusions:}

MALDI-MS analysis can be used to distinguish isobaric cross-linked
peptides with thiazine and thiosuccinimide linkers. Peptides that are
cross-linked with thiazine linkers are very stable in MALDI and are thus
detected only as (M+H)\textsuperscript{+} ions. On the other hand,
cross-linked peptides with thiosuccinimide as the linker are clearly
more susceptible to degradation by MALDI, and the thioether bond of
thiosuccinimide is partially cleaved. As a result, in addition to the
intact cross-linked peptides, the free thiol-containing peptides and
peptides attached to the original \emph{N} -propionyl succinimide, which
constitute the conjugate, are clearly detected in the MALDI-MS spectra.
This behavior was also observed for cross-linked peptides with
hydrolyzed thiosuccinimide linkers.

MALDI-MS/MS analysis can more reliably distinguish isobaric cross-linked
peptides with thiazine and thiosuccinimide as linkers. The former gives
fragment ions derived from cleavage at the new pseudo-peptide bond
produced by trans-cyclization in the thiosuccinimide
linker\textsuperscript{14,15}; the latter gives two types of the
fragment ions due to 1,4-elimination that occurs in the gas phase
(Figure 4). MALDI-MS or MALDI-MS/MS can be used as a simple technique to
identify isobaric cross-linked peptides that are prepared via maleimide
chemistry.

\textbf{Figures Legends:}

\textbf{Figure 1} . Possible cross-linking structures between Cys and
Lys prepared by thiol-maleimide chemistry. Structure \textbf{(I)} :
thiosuccinimide form; \textbf{(II), (III)} : Hydrolyzed forms
of\textbf{(I)} , in which the five-membered thiosuccinimide ring was
cleaved at ``a'' or ``b'', respectively; \textbf{(IV)} : thiazine form,
which is formed by the attack of the \selectlanguage{greek}α-\selectlanguage{english}amino group of Cys on the
carbonyl of the succinimide moiety and has the same molecular mass
as\textbf{(I)} . The dashed square in Structure IV shows the new
pseudo-peptide bond present in the thiazine ring.

\textbf{Figure 2.} Reverse-phase liquid chromatograms of the P1-P2
cross-linked peptide obtained after the synthesis \textbf{(b)} and
incubation in 100 mM Tris/HCl buffer, pH=8.0 for 6 hours at
37\textsuperscript{o}C \textbf{(a)} . The amino acid sequences of P1 and
P2 are (\emph{A} AAGGGAAAAK) and (\emph{C} AAGGGAAAAK), respectively.
The peaks marked with asterisks in both chromatograms correspond to the
disulfide-linked P2-S-S-P2 homodimer formed as a by-product. The left
and right Y-axes represent the absorbance (mV) at 210 nm of the upper
and lower chromatograms, respectively. Numbers in red above each
fraction indicate retention times.

\textbf{Figure 3.} MALDI-MS spectra of the cross-linked peptide, P1
(\emph{A} AAGGGAAAAK)-P2 (\emph{C} AAGGGAAAAK)\textbf{(a)} , its
hydrolyzed (P1-P2)\textsubscript{H} \textbf{(b)} and thiazine
(P1-P2)\textsubscript{T} \textbf{(d)} forms. The insets in \textbf{(a)}
and\textbf{(b)} are the expanded views of the red dashed squares. The
signals marked with I, II, III, and IV in the insets are assigned to the
structures in \textbf{(d)} . The structures of P1-P2,
(P1-P2)\textsubscript{H} and (P1-P2)\textsubscript{T} are depicted at
the right side in \textbf{(a)} , \textbf{(b)} and \textbf{(c)} ,
respectively. P2-SH in the spectra correspond to the P2 peptide with a
free Cys residue.

\textbf{Figure 4.} The 1,4-elimination observed for the cross-linked
peptide containing thiosuccinimide. The two possible pathways are
depicted in \textbf{(a)} and \textbf{(b)} . P\textsubscript{n} denotes
the peptides used in this study.

\textbf{Figure 5} . MALDI-MS spectra of the cross-linked peptides, P2
(\emph{C} AAGGGAAAK)-P4 (Ac-GANAP\emph{K} EPQR) (a) and the thiazine
form (P2-P4) \textsubscript{T} (b). The inset of (a) is the expanded
view of the red dashed square. P2-SH corresponds to the P2 peptide with
a free Cys residue. The signals marked with P4* in (a) correspond to the
structures depicted in (c).

\textbf{Figure 6} . MALDI-MS/MS spectra of the cross-linked peptides,
P1-P2 (a) and (P1-P2)\textsubscript{T} (d). The areas enclosed by the
dashed squares in (a) and (d) are enlarged in (b) and (c), respectively.
The \emph{m/z}values in green and red indicate fragment ions that are
specifically observed for the thiosuccinimide and thiazine forms,
respectively. ``L'' indicates the \emph{N} -propionyl maleimide linker.
The assignments of backbone-derived fragment ions are summarized in
Figures S10-S11.

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\textbf{CRediT Classification:}

\textbf{LJG} : Conceptualization, Formal Analysis, Writing -- Original
Draft Preparation, Visualization, Investigation. \textbf{SP} : Writing
-- Original Draft Preparation, Visualization, Investigation, Formal
Analysis. \textbf{HH} : Resources, Investigation, Writing -- Review \&
Editing. \textbf{SW} : Investigation, \textbf{DH} :
Investigation,\textbf{ARM} : Funding Acquisition. \textbf{TT} : Formal
Analysis, Conceptualization, Resources, Funding Acquisition,
Supervision, Writing -- Review \& Editing.\selectlanguage{english}
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