Mass Spectrometry Analysis of Daunorubicin-Peptide Drug Conjugates

This report focuses on the mass spectrometry analysis of peptide drug conjugates, specifically examining daunorubicin-containing bioconjugates. The study explores the impact of structural modifications, such as the number of charged functional groups and the distance between daunomycin and the peptide, on gas-phase stability and fragmentation patterns. The research details the synthesis of peptides using solid-phase peptide synthesis and the conjugation of daunomycin. The report also discusses the application of MALDI-TOF/TOF mass spectrometry for characterizing peptide bioconjugates, including the analysis of stereoselective disintegration and the sequence dependence of fragmentation patterns. Experimental procedures, including peptide synthesis and MALDI-TOF/TOF mass spectrometry techniques, are described. Furthermore, the report provides an overview of mass spectrometry principles, ionization sources, mass analyzers, and tandem mass spectrometry, emphasizing the importance of MS/MS for structural analysis and quantification of molecules in various fields, including biotechnology, clinical testing, and pharmaceutical analysis. The report also covers the use of LC-MS/MS in the analysis of peptide bioconjugates and the advantages of mass spectrometry over traditional methods like ELISA.

MASS SPECTROMETRY ANALYSIS FOR PEPTIDES DRUG
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Energy set on MS/MS disconnection of daunorubicin-peptide conjugates
This research was keen on detecting daunomycin-containing peptide conjugates and
determining suitable circumstances for the bulk spectrometric classification of these
composite molecules. Due to this reason, new tuftsin-based bioconjugates were synthesised
to examine the gas-phase ability of dau as the positively charged amino acid remains existed.
The structural elements influence on the fragmentation study was conducted in details. It
included;
Figures of drug molecules
Figures of elementary groups that were functioning (Langel, 2014, p. 44)
Existence or non-existence of a broadly applied enzyme-labile spacer (GFLG) amid the
peptide targeted and the molecule of the drug. The expectations were that these structured
modifications, i.e. the decreasing of the figures of the functional groups that were charged
and the distance increased between the Dau and the peptide, would change the phase of the
gas’s stability of daunomycin and the fragmentation could not be the same. In addition, the
key goal was to overwhelm the in-source losses in sugars, and therefore detect protonated

molecules that are intact. It was aimed, therefore, in optimizing the mass spectrometric
conditions, solvents and also source of iron parameters.
Daunomycin-Peptide Bioconjugates preparation
Peptides were manually synthesized by solid-phase peptide synthesis through application of
Fmoc tactic on Wang resin (0.6 mmol/g capacity) was attached using the same DIC amount
in N,N dimethyformamide in 0.1 equiv. presence TO THE derivative amino acid. The
remaining amino acids were attached with DIC/HOBt in DMF. Fmoc collection was chopped
with 2 percent piperidine and 2 percent DBU in DMF in four stages (2+2+5+10 min). Free
N-terminus peptides were synthesised through Boc-(Thr)- OH as the peptide backbone’s last
amino acid. (Frier & Perkins, 2016, p. 144)
The 2, 4, 6 trichlorophenyl formate was reacted with the N-terminal of formylated
derivatives. Thereafter, the side chain lysine protecting group was cleaved selectively with
2% hydrazine hydrate in DMF on resin, and Boc-protected aminooxyacetic acid was either
coupled directly to the amino group of the lysine or to a GFLG spacer which was earlier built
on the side of the lysine with the standard Fmoc practice.
Peptides were chopped from the resin, and were all groups of protection were removed with
95 percent trifluoroacetic acid, 2.5 percent water, 2.5 percent TIS (v/v/v) in the existence of
10 equiv. aminooxyacetic acid free, as carbonyl capture for 30 minutes at 0 degrees reagent ,
then 2 hours room temperatures followed by ice-cold diethyl ether precipitation, washing
with diethyl ether and solubilisation 3 times in a water that is distilled for freeze-drying.
These crude peptides were disinfected by RP-HPLC, and the purified fractions were used
immediately for the following synthetic stage after solvent evaporation. Daunomycin was
conjugated to the peptides in the solution at a concentration of 10mg/ml peptide. (Dumitriu,
2013, p. 234)

The mixture reactions were, for about 16 hours, stirred at a room temperature. The resulted
bioconjugates were disinfected by RP-HPLC. The conjugates purity was examined by
analytical HPLC by applying Knauer HPLC system and a C18 column Nucleosil. 0.1 percent
TFA in water eluents and 0.1 percent TFA in acetonitrile-water. The rest of separations of
chromatography were conducted at a room temperature. (Ryadnov & Hudecz, 2017, p. 181)
Techniques of MALDI/TOF/TOF for categorization of peptide bioconjugates
Bioactive peptides are engaged in various functions biologically, and serve key roles as
hormones and neuropeptides for cellular signalling, as secretory peptides for interspecies
message, or as peptide toxins and also defence peptides against microbes and predatory
animals.
For the comprehension of all these methods, analysis and knowledge in their primary
structure is important. As a result, an array and enough methods like analysis of amino acids
or sequencing of tandem MS is available, which permits for the determination routine of
amino acid sequences and the major modifications of chemicals.
However, some peptides carry along a perfidiously naïve post-translational modification that
is not detected immediately by the standard analytical measurements. In some proteins and on
the peptides, the dogma of ubiquitous protein homochirality is abused by one D-amino acid
replacement. (Aslam & Den, 2016, p. 210)
In some circumstances, a D- remains in peptide bond can come from age-dependent
racemization. Though, throughout peptide biosynthesis, which is totally unconnected to
getting old, the D- amino acid is produced from the equivalent L-isomer existing within the
precursor polypeptide by the action of peptidyl-aminoacyl-L/D-isomerases. Amusingly,
all L/D-isomerases considered to date from vertebrates act entirely on the second N-terminal

amino acid remains. The initial peptide, which was originated to be treated by an enzyme,
was dermorphin sequestered from the skin of a South American tree frog. Additionally, the
finding of dermorphin and also the linked deltorphinswas simplified by the remarkable
outcome of the D- remains on biological goings-on (i.e., the all-L peptides were inactive). By
comparing, more subtle effects were witnessed in other vertebrate peptides, in which an
ordinary form with a D- amino acid happens as well, for example, the bombinins H from th
skin of the frog, and, amusingly, a C-type natriuretic peptide and a β-defensin-like peptide
from the venom of male platypus, a simple mammal. (Wang, et al., 2016, p. 108)
We systematically discovered the sequence reliance of the stereoselective disintegration
tendency in MALDI-TOF/TOF metastable decay–CID experiment on the study. We
dedicated on model peptides with a single D- amino acid remains in position 2 as peptides
bearing a reverse of backbone chirality at this spot might be of uppermost biological
importance in vertebrates. We examined the outcome of different D- remains in position 2 as
well as the modifying effect of the nearest neighbor remains on the pattern of fragmentation.
Our data recommend that the stereosensitivity is extremely sequence-dependent and can be
certainly of real-world use for the scrutiny of a verity of peptide sequences. As a chief
experimental development, the examination of stereoisomers with unfavorable R became
feasible by spending a deuterated analogue of the doubted epimeric candidate. As a proof-of-
concept, we were able to settle on the existence of a D- remains in a projected peptide from
frog skin emissions by means of SF-MALDI-TOF/TOF MS. (Yadav, 2016, p. 98)
Experimental processes
Peptide synthesis
Peptides were blended on a continuous-flow synthesizer by means of standard Fmoc solid-
phase chemistry with a Rink Amide AM resin (200–400 mesh, 0.62 meqg–1; Nova-Biochem),

PyBOP (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate) as
condensation reagent, and N-methylmorpholine as a base. After cleavage, peptides were
derivatized with iodoacetamide and refined by reverse-phase high-performance liquid
chromatography (HPLC) over a C-18 column (Vydac) with a linear gradient of acetonitrile
(solvent A, 0.1% TFA; solvent B, 80% acetonitrile). N-Fmoc-Val-d8 (N-(9-
fluorenylmethoxycarbonyl)-L-valine-2, 3, 4, 4, 4, 5, 5, 5-d8) was from Aldrich. (Toniolo,
2018, p. 330)
MALDI-TOF/TOF mass spectrometry
Trials were examined on a MALDI-TOF/TOF 4800 analyser, ABSciex Framingham, worked
in reflector positive mode obtaining 3000 total shots per spectrum with a fixed laser
concentration set at 5000. Experiments were approved out using α-cyano hydroxyl cinnamic
acid (5 mg/mL in 50% (v/v) acetonitrile) as matrix. Every sample (0.3–0.5 μL) was mixed
with 2–3 μL of matrix, and 0.5 μL of the combination was spotted on the objective plate.
Tandem MS experiments were approved out using a 1 kV method with and without collision-
induced disconnection using air as collision gas and metastable suppression enabled. Spectra
were developed and processed using the 4800 Analyzer and Data Explorer Software. In most
circumstances, when water loss peaks were detected, peak areas a were united with their
parent ion peaks. Likened to PSD, the application of CID favored various fragmentations and,
in such a way, problematical analysis of data. (Birch & Lennox, 2015, p. 204)
R matrices were produced for every compound, agreeing to formulas Ri,j = (ri,j)L/(ri,j)D and ri,j =
(ai/aj). Thus, a are the peak areas of two selected fragment ions i and j in a spectrum of
epimer L or D, correspondingly. ri,j were averaged from three spectra, each of a unlike spot.
For practical reasons, these matrices can be scrutinized for extreme values without additional
processing.

Peptide GH-2 isolation
Secretion of skins were collected from Bombina orientalis . They were defined and went
through a Centricon filter holding proteins with a mass of >10 kDa. The filtrate was
fractionated over a 218TP C-18 column (Vydac, Hesperia, CA) with a gradient of acetonitrile
(solvent A, 0.1% TFA; solvent B, 80% acetonitrile in solvent A). Fractions yielding a mass
peak corresponding with the deliberated mass of GH-2 peptide were dried out in the
SpeedVac, exposed to endoproteinase Asp-N (Sequencing grade, Sigma) cleavage in 100
mM NH4HCO3buffer, pH 8.5, and were rechromatographed under the similar conditions.
(Brown, 2014, p. 45)

References
Aslam, M. & Den, A., 2016. Bioconjugation: protein coupling techniques for the biomedical
sciences. 3rd ed. London: Macmillan Reference.
Birch, J. R. & Lennox, E. S., 2015. Monoclonal Antibodies: Principles and Applications. 3rd
ed. Texas: Wiley.
Brown, D. M., 2014. Drug Delivery Systems in Cancer Therapy. 4th ed. Chicago: Springer
Science & Business Media.
Dumitriu, S., 2013. Polymeric Biomaterials, Revised and Expanded. 2nd ed. Chicago: CRC
Press.
Frier, M. & Perkins, A. C., 2016. Nuclear Medicine in Pharmaceutical Research. 3rd ed.
Texas: CRC Press.
Langel, Ü., 2014. CPP, Cell-Penetrating Peptides. 4th ed. Texas: Springer.
Ryadnov, M. & Hudecz, F., 2017. Amino Acids, Peptides and Protein. 4th ed. London: Royal
Society of Chemistry.
Toniolo, C., 2018. Peptaibiotics. 3rd ed. Texas: John Wiley & Sons.
Wang, J., Shen, W. C. & Zaro, J. L., 2016. Antibody-Drug Conjugates. 3rd ed. Chicago:
Springer.

Yadav, S. K., 2016. Nanoscale Materials in Targeted Drug Delivery, Theragnosis and Tissue
Regeneration. 5th ed. London: Sudesh Kumar Yadav.