Conjugation Site Analysis by MS/MS Protein Sequencing

In-depth knowledge of drug-linker conjugation sites is very important for antibody-drug conjugate (ADC) structural elucidation at both drug discovery and development stages. Peptide mapping has proven itself to be an effective tool for the determination of conjugation sites by site-specific protein cleavage with a proteolytic enzyme, followed by gradient elution of the resulting peptides through reverse-phase liquid chromatography (RPLC).

This article describes a peptide mapping-based protocol to determine the locations of drug-linker attachment on mAbs, aiming to provide researchers with a quick and easy approach to characterizing and optimizing ADCs.

Disclaimer

This procedure is only a guideline. Please note that Creative Biolabs is unable to guarantee experimental results if it is operated by the customer.

• Digested Peptides Sample Preparation

Materials:
100 mM tromethamine hydrochloride (Tris–HCl), pH 7.8 buffer.
Denaturing Buffer: 8 M urea in 100 mM Tris–HCl, pH 7.8 buffer.
500 mM dithiothreitol (DTT).
500 mM iodoacetamide (IAM).
1 mg/mL lyophilized trypsin (see Note 1).

Procedure:
1. Dilute the sample to 1 mg/mL in 500 μL of denaturation buffer in a polypropylene tube (see Note 2).
2. Add 5 μL of 500 mM DTT to the above tube, and then mix the contents of the tube gently (see Note 4).
3. Incubate the tube at 37 °C for 30 min in the dark (see Note 4) and then cool it down to room temperature.
4. Add 10.2 μL of 500 mM IAM to the above tube, then mix the contents of the tube gently (see Note 5), and incubate the tube at room temperature for 30 min in the dark.
5. Take 100 μL of the reduced and alkylated sample for buffer exchange through spin desalting columns, and then collect the sample and place it in 100 mM Tris-HCl, pH 7.8.
6. Measure the protein concentration of the buffer-exchanged sample at 280 nm. Use 100 mM Tris–HCl, pH 7.8 solution as blank for baseline correction (see Note 6).
7. Calculate the volume needed for 50 μg of buffer-exchanged sample and transfer this volume to a new polypropylene tube.
8. Add acetonitrile to the sample to a final concentration of 10% (v/v), and mix the contents of the tube gently (see Note 7).
9. Add a certain volume of the above trypsin to the 50 μg aliquot of the sample that has been added with 10% acetonitrile to achieve an enzyme : substrate (E:S) ratio (w/w) of 1:25–1:18. Mix the contents of the tube gently and incubate the sample at 37 °C for 1–2 h in the dark (see Note 8).
10. Add isopropanol to the sample to a final concentration of 40% (v/v), and mix the contents of the tube gently (see Note 9).
11. The above-resulting samples are stored at -80 °C for subsequent analysis (see Note 10).

Note:
1. Trypsin is the first-choice enzyme for peptide mapping analysis of mAbs and ADCs, which specifically cleaves at the carboxylic side of lysine and arginine residues.
2. 8 M urea is preferred over 6 M guanidine chloride (GndHCl) as the denaturing buffer because 8 M urea provides a stronger denaturing condition, which enables proteolytically resistant proteins to be digested more efficiently.
3. It is recommended to use DTT rather than tris(2-carboxyethyl)phosphine (TCEP) as the reducing agent because TCEP can reduce oxidized methionine residues if already present on the molecule before digestion.
4. The recommended temperature condition for reduction is 37 °C. This can prevent protein carbamylation, which occurs in urea solutions at high temperatures (>60 °C).
5. It is recommended to use IAM rather than iodoacetic acid (IAA) as the alkylating agent due to its faster reaction time, and unlike IAA, it does not introduce a negative charge to the derivatized peptide.
6. It is strongly recommended to use a micro-volume spectrophotometer so that the majority of the sample remains untouched from the auto-pipette and cuvette to avoid sample contamination and degradation.
7. In order to maintain hydrophobic drug-linker loaded peptides in solution such that all the conjugated peptides can be identified on the LC-MS chromatogram, 10% acetonitrile is suggested to be added to the sample prior to digestion.
8. The enzyme is suggested to be added more than conventional use (E:S ratio of 1:50) to shorten the digestion time and minimize the mAb in-digestion modification (asparagine deamidation or N-terminal glutamine cyclization). If the drug linker is thermally unstable, the incubation can be conducted at room temperature for 4 h in the dark.
9. It is suggested that 10% acetonitrile be added to the sample before digestion and 40% isopropanol after digestion to keep the hydrophobic drug-linker loaded peptides in solution after enzymatic digestion.
10. It is strongly recommended to make fresh-digested peptide samples right before the LC-MS experiment for stability and solubility of drug-linker loaded peptides.

• Digested Peptides Sample Separation

Materials:
Ultra high performance liquid chromatography (UHPLC) (see Note 11).
Octadecylsilane (C18) column with 300 A or smaller pores and particle size of 2–3 μm or less.
Mobile phase A: LC-MS grade water with 0.085% FA, 0.015% TFA.
Mobile phase B: LC-MS grade acetonitrile with 0.085% FA, 0.015% TFA.

Procedure:
1. Set up the flow rate at 0.1–0.2 mL/min.
2. Set up the following elution gradient:
0% MPB for 4 min, 0–65% MPB for 60 min, 65–100% MPB for 10 min, 100% B for 5 min, 100–0% B for 1 min, 0% B for 10 min (see Note 12).
3. Set up the ultraviolet (UV) detection wavelength are 214 nm and the λ_max of the drug-linker (see Note 13).
4. Set up MS detection parameters:
An electrospray ionization (ESI) source operated in positive mode is used to introduce positively charged peptide ions.
Acquire the MS scan with a mass/charge (m/z) range of 50–2500, followed by MS/MS (tandem MS) scans of precursor ions (see Note 14).

Note:
1. It is strongly recommended to use the UHPLC system as it significantly enhances the efficiency of LC using the columns with sub-2-μm particle size and UHPLC systems with low dead volume and tolerance of up to 15,000 psi.
2. Given the hydrophobic properties of drug-linker loaded peptides, it is sometimes needed to elute them by using a higher percentage of mobile phase B.
3. It is strongly recommended for detection at 214 nm (i.e., a wavelength where peptide bonds absorb light) and the use of dual wavelength by including the λ_max of the drug-linker as the second UV channel to readily identify the peaks of the drug-linker-loaded peptides in the UV chromatogram during data analysis.
4. The LC-MS/MS scan of the peptide fragmentation ions is very useful for the characterization of drug-linker loaded peptides, which can be used to recognize conjugated peptides in the LC-MS/MS map and allow for the identification of conjugation sites without ambiguity, even in situations where multiple modification sites are present in a single digested peptide.

• Data Analysis

Procedure:
You can choose either of the following two methods:
A. Software Searching Algorithm
Input the experimental data and light chain and heavy chain sequence in the searching engine software, set the enzyme as trypsin, select the fragmentation type, and input the common PTMs (N-linked glycosylation, deamidation (Asn, Gln), oxidation (Met, Trp, Cys), N-terminal pyroglutamate formation, or C-terminal Lysine truncations), as well as the mass shift caused by drug-linker conjugation as modifiers. Set the mass tolerance for precursor ions and fragment ions (typically ± 5 ppm) and the intensity threshold as filters. Once completed, the search algorithm identification for drug-linker-loaded peptides can be performed (see Note 15).
B. Manual Data Review
1. For interchain cysteine-conjugated ADCs (native or engineered), calculate the expected mass of all the putative, uncleaved tryptic peptides. Then search in the LC-MS profile for these masses by plotting extracted ion chromatograms (EICs) (see Note 16).
2. Use the characteristic MS/MS fragment ions originating from the payload as signature ions for quick identification of conjugation sites without ambiguity.
3. Use the chromatogram by UV detection at the λ_max to identify conjugated peptides.

Note:
1. In principle, peptide identification by searching engines relies on both peptide mass measurement and fragmentation products observed in MS/MS spectra. However, in certain cases, the MS/MS scan of drug-linker-loaded peptides through CID fragmentation may result in inadequate fragmentation of the peptide backbone. Depending solely on MS1 data can result in incorrect positive identifications or missing information due to limitations imposed by the software filter setting. So, manual data review is necessary to confirm the assignments.
2. This step can help quickly confirm the characterization of the expected drug-linker-loaded peptides linked to the inter-chain cysteine of the HC and LC. However, it cannot work very efficiently for lysine-conjugated and cysteine-conjugated ADCs. Under such circumstances, the characteristic MS/MS fragment ions are needed to identify the unexpected, nonspecific drug-linker conjugation sites.

Creative Biolabs can provide comprehensive ADC binding site analysis and ADC structure analysisservices for clients to help them identify the most suitable candidate drugs.