Protocol for structural modeling of antibody to human leukocyte antigen interaction using discovery and targeted cross-linking mass spectrometry

Summary

Cross-linking mass spectrometry (XL-MS) provides low-resolution structural information to model protein structures. Here, we present a protocol to identify cross-links of purified antibody binding to purified human leukocyte antigen (HLA). We describe steps for using a discovery-based XL-MS approach followed by a targeted XL-MS approach. We then detail procedures for using the identified cross-links with other structural data for molecular docking of the antibody to HLA. This protocol has applications for modeling the interacting structure of purified antibody to antigen.

For complete details on the use and execution of this protocol, please refer to Ser et al.¹

Graphical abstract

Highlights

• •Protocol for cross-linking mass spectrometry for antibody to human leukocyte antigen
• •Discovery and targeted XL-MS procedure for confident cross-link identification
• •Procedure for molecular docking with XL-MS input to model antibody to HLA structure

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Cross-linking mass spectrometry (XL-MS) provides low-resolution structural information to model protein structures. Here, we present a protocol to identify cross-links of purified antibody binding to purified human leukocyte antigen (HLA). We describe steps for using a discovery-based XL-MS approach followed by a targeted XL-MS approach. We then detail procedures for using the identified cross-links with other structural data for molecular docking of the antibody to HLA. This protocol has applications for modeling the interacting structure of purified antibody to antigen.

Before you begin

Cross-linking mass spectrometry (XL-MS) provides low resolution structural information that can be used in integrative structural modeling to model protein structures.² Both predicted structural information and experimental information can be used for molecular docking of interacting proteins.³ Here, we describe a technique that uses discovery and targeted XL-MS acquisition of purified antibody (Ab) and purified human leukocyte antigen (HLA).¹ The cross-link data can be used as a distance restraint for molecular docking of Ab to HLA, together with protein structures of Ab, HLA and predicted interacting surfaces of Ab and HLA. The obtained structural model can be used to understand the molecular basis of antibody to HLA interaction.

Before initiating the experiment, prepare purified antibody and purified human leukocyte antigen protein. Sequences of the antibody and HLA proteins in fasta format are required for the database searching of the mass spectrometry data and for predicting antibody structure and interacting residues with HLA protein for molecular docking. Predicted eplets of human leukocyte antigen are required as interacting residues for molecular docking of antibody to HLA structure.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Dithiothreitol	Bio Basic	Cat# DB0058
Phosphate-buffered saline (PBS)	Biowest	Cat# L0615-500
Sulfuric acid	Sigma-Aldrich	Cat# 32050-1
SDA (NHS-Diazirine) (succinimidyl 4,4′-azipentanoate)	Thermo Scientific	Cat# 26167
Disuccinimidyl sulfoxide	Thermo Scientific	Cat# A33545
Dimethyl sulfoxide	Sigma-Aldrich	Cat# 276855
Tris(2-carboxyethyl)phosphine (TCEP)	Goldbiochem, Axil Scientific	Cat# TCEP10
Chloroacetamide	Sigma-Aldrich	Cat# C0267-100G
Triethylammonium bicarbonate (TEAB), 1 M, pH 8.5	Merck	Cat# T7408
Lysyl endopeptidase (LysC)	Fujifilm Wako Chemicals	Cat# 129-02541
Trypsin protease, MS grade	Pierce	Cat# 90058
Trifluoroacetic acid, MS grade	Sigma-Aldrich	Cat# T6508-100ML
Water (LC-MS grade)	Merck	Cat# W6-4
Water with 0.1% formic acid (LC-MS grade)	Merck	Cat# LS118-4
Acetonitrile (LC-MS grade)	Merck	Cat# A955-4
Acetonitrile with 0.1% formic acid (LC-MS grade)	Merck	Cat# LS120-212
Software and algorithms
MetaMorpheus v0.0.318	Lu et al.4	https://github.com/smith-chem-wisc/MetaMorpheus
Skyline v21.2.0.568	Pino et al.5	https://skyline.ms/project/home/begin.view
Cross-link Transition Calculator	Chavez et al.6	https://skyline.ms/skyts/home/software/Skyline/tools/details.view?name=Cross-link%20Transition%20Calculator
ABodyBuilder	Leem et al.7	http://opig.stats.ox.ac.uk/webapps/abodybuilder
Antibody i-Patch	Krawczyk et al.8	https://opig.stats.ox.ac.uk/webapps/sabdab-sabpred/sabpred/more#Antibody%20i-Patch
HADDOCK 2.4	de Vries et al.9	https://wenmr.science.uu.nl/haddock2.4/
PyMOL v2.5.5	Schrodinger	https://pymol.org/2/
Other
Oasis HLB 10 mg cartridge	Waters	186000383
EASY-Spray column	Thermo Scientific	ES903
HiLoad 16/600 Superdex 200 column	GE Healthcare Life Sciences	GE28-9893-35

Step-by-step method details

Cross-linking of purified antibody to purified human leukocyte antigen

Timing: 2–3 h

Purified antibody and purified human leukocyte antigen are chemically cross-linked

• 1.Add purified antibody (20 μg) to purified HLA protein (12 μg) in 45 μL of PBS buffer and mix. Antibody to HLA molar ratio is 1:2.

Note: Approximately 20–50 μg of starting protein is needed to ensure sufficient cross-linking mass spectrometry intensity downstream. Full-length IgG antibody to HLA molar ratio of 1:1 and 1:2 have minimal impact on identified cross-links (unpublished data). Use of antibody fab fragment instead of full-length IgG antibody also returns similar results for identified cross-links (unpublished data). See troubleshootingproblem 1 and problem 2.

• 2.Let antibody, HLA proteins sit at 25°C for 15 min to allow proteins to interact.

CRITICAL: Purified antibodies and purified HLA protein should be in amine-free buffer such as PBS. Presence of amines will quench any cross-linking activity of amine-reactive cross-linkers. Purified proteins can be buffer exchanged into PBS buffer using 30 K MWCO protein concentrator.

Note: For cross-linking of proteins using disuccinimidyl sulfoxide, DSSO, cross-linker, proceed with steps 3–6. DSSO is a MS-cleavable cross-linker that targets lysine-to-lysine residues, with side-reactivity for serine, threonine and tyrosine residues. DSSO has a spacer arm of 10.1 Å. Alternative lysine-to-lysine reactive cross-linkers are disuccinimidyl glutarate, DSG with a spacer arm of 7.7 Å and carbonyldiimidazole, CDI with a spacer arm of 2.6 Å.

• 3.Prepare 100 mM DSSO by dissolving 1 mg of DSSO powder in 25.75 μL of DMSO. Aspirate using a pipette to ensure that DSSO has dissolved.
• 4.Add 5 μL of 100 mM DSSO to antibody, HLA proteins. Gently aspirate using pipette to ensure proper mixing of cross-linker with proteins.
• 5.Incubate proteins with cross-linker for 60 min at 25°C.
• 6.Add 5 μL of 1 M Tris, pH 8 and incubate for 15 min at 25°C to quench cross-linking.

Note: For cross-linking of proteins using succinimidyl-diazirine, SDA cross-linker, skip steps 3–6 and proceed with steps 7–12. Steps 7–11 have to be performed with minimal exposure to light. SDA is activated by UV-light and targets lysine-to-any amino acid residue. The succinimidyl end has reactivity with lysine and side-reactivity for serine, threonine and tyrosine, while the diazirine end has reactivity with any amino acid with some preference for aspartate and glutamate residues. We recommend using a second cross-linker in addition to lysine-to-lysine reactive cross-linkers to improve the chance of identifying cross-links between antibody to HLA. SDA has a spacer arm of 3.9 Å. An alternative lysine-to-any reactive cross-linker is LC-SDA with a spacer arm of 12.5 Å.

• 7.Prepare 100 mM SDA by dissolving 1 mg of SDA powder in 44.4 μL of DMSO. Aspirate using a pipette to ensure that SDA has dissolved.
• 8.Add 5 μL of 100 mM SDA to antibody, HLA proteins. Gently aspirate using pipette to ensure proper mixing of cross-linker with proteins.
• 9.Incubate proteins with cross-linker for 60 min at 25°C in the dark.
• 10.Add 5 μL of 1 M Tris, pH 8 and incubate for 15 min at 25°C to quench NHS-ester cross-linking.
• 11.Position purified proteins and Eppendorf tubes within 3–5 cm of a UVP lamp. Turn on UVP lamp (8 W) at 365 nm to photo-crosslink proteins. Incubate proteins for 60 min under the UVP lamp.

CRITICAL: Intensity of UV light for photo-crosslinking is dependent on power of UVP lamp used, distance of proteins from the UVP lamp and length of time to UV exposure.

• 12.Add 5 μL of 1 M Tris, pH 8 and incubate for 15 min at 25°C. Cross-linked proteins can be exposed to light at this step.

Pause point: Cross-linked proteins can be stored at -80°C for a few days. We do not recommend long-term storage of cross-linked proteins as sensitivity of cross-linking mass spectrometry may be decreased by long-term storage.

• 13.Confirm cross-linked proteins by running aliquots of cross-linked protein on a SDS-PAGE gel and perform staining.

Note: High molecular weight bands corresponding to the combined molecular weight of the antibody and HLA will indicate successful cross-linking of antibody to HLA. See Figure 4A for example of stained protein gel for successful cross-linking of proteins.

CRITICAL: We recommend assessing completeness of cross-linking before proceeding to mass spectrometry. While not all proteins are expected to be cross-linked, a majority of proteins should appear as a higher molecular weight band as an indication of successful cross-linking.

Note: A 5 μL aliquot of cross-linked proteins (after step6 or after step12) can be taken to confirm cross-linking by SDS-PAGE gel. Mix 5 μL aliquot of cross-linked proteins with 4× LDS sample buffer with 2 mM DTT and top off volume to 20 μL with MilliQ water. Heat proteins at 95°C for 10 min to denature proteins and leave to cool. Load proteins on a 4–12% Bis-Tris Gel and run with MES running buffer at constant 120 V for 30 min. Stain gel with Coomassie G-250 staining solution for 1 h. Leave to destain in MilliQ water overnight. Observation of high molecular weight bands corresponding to the combined molecular weight of antibody and HLA will indicate successful cross-linking of antibody to HLA.

Figure 4.

Example results of cross-linking data from discovery XL-MS, targeted XL-MS and molecular docking

(A) Example Coomassie stained SDS-PAGE gel for confirmation of DSSO and SDA cross-linked antibody-HLA complex. Gel bands: 1) ∼150–200 kDa, Cross-linked antibody-HLA complex. 2) 55 kDa, 2E3 antibody heavy chain. 3) ∼32 kDa, HLA α-chain. 4) 25 kDa, 2E3 antibody light chain. 5) 12 kDa, HLA β-chain.

(B) Discovery XL-MS identification of cross-links from 2E3 antibody to HLA-A∗11:01 (C) Extracted chromatograms of targeted XL-MS peaks for inter-link between 2E3 antibody Y52 to HLA-A∗11:01 K68 (D) Crystal structure (PDB ID: 6ID4) and molecular docking structures based on restraints (E) with cross-link input and without interacting surface input, (F) with cross-link input and interacting surface input, and (G) without cross-link input and with interacting surface input. Figure modified and reprinted with permission from Ser et al. (2023).

Sample preparation of cross-linked proteins for mass spectrometry

Timing: 2 days

Cross-linked proteins are reduced, alkylated, digested and desalted and ready for analysis by liquid chromatography mass spectrometry.

• 14.Add 1 μL of 500 mM TCEP to cross-linked proteins and incubate for 30 min at 25°C to reduce proteins.

Alternative: Dithiothreitol, DTT, can be used as a reducing agent instead of TCEP. DTT is more susceptible to degradation, so should be prepared fresh before use.

• 15.Add 5 μL of 550 mM CAA and incubate for 30 min at 25°C in the dark to alkylate proteins.
• 16.Add 100 μL of 100 mM TEAB, pH 8.5.
• 17.Add 2 μL of 0.5 μg/μL LysC and shake on a thermomixer at 600 rpm for 4 h at 25°C.

Note: Addition of LysC protease should help to pre-digest cross-linked protein and increase accessibility of lysine and arginine for further digestion by trypsin.

• 18.Add 2 μL of 0.5 μg/μL trypsin and shake on a thermomixer at 600 rpm for 18 h at 25°C.
• 19.Add 20 μL of 10% TFA to acidify solution to quench protease digestion.

Optional: Take 1 μL of acidified solution and test pH using a pH strip. Solution should be pH 2-3 to ensure that samples are at the acidic pH for proper retention and elution of cross-linked peptides by desalting in Step 19.

• 20.
  Desalt digested cross-linked proteins using an Oasis HLB 10 mg cartridge

  • a.
    Activate Oasis HLB 10 mg cartridge by passing 200 μL of ACN. Discard flowthrough.
  • b.
    Equilibrate by passing 200 μL of 0.1% formic acid in water through cartridge twice. Discard flowthrough.
  • c.
    Load digested cross-linked proteins and pass through the cartridge.
  • d.
    Wash by passing 200 μL of 0.1% formic acid in water through cartridge twice. Discard flowthrough.
  • e.
    Elute by passing 200 μL of 65% ACN, 0.1% formic acid in water through cartridge. Collect elution in a new Eppendorf tube.

Alternatives: Desalting can be performed using other commercially available C18 cartridges, e.g. Pierce C18 spin columns.

• 21.Dry eluted peptides by vacuum centrifugation. Store dried peptides at -20°C.

Pause point: Dried peptides can be stored at -20°C for a few weeks.

Discovery cross-linking mass spectrometry and data analysis to identify cross-linked peptides

Timing: 1–2 days

Cross-linking mass spectrometry performed in Data-dependent acquisition (DDA) for discovery of cross-linked peptides from antibody to HLA.

• 22.Resuspend dried peptides to 0.5 μg/μL concentration with 64 μL of buffer (2% ACN, 0.06% TFA, 0.5% acetic acid in water).

Note: Dried peptides are resuspended to 0.5 μg/μL concentration to load 2 μL or 1 μg on an Easy-Spray column for LC-MS acquisition. Approximately 1 μg of peptide material should be loaded on the LC-MS column to prevent over or under-loading.

• 23.Centrifuge at 20,000 g for 10 min to pellet any particles or aggregates. Transfer supernatant to the load plate for LC-MS acquisition.

Note: Particles or aggregates may result in clogging of column and overpressure of LC system. Centrifugation to remove particles or aggregates will improve long term LC-MS performance.

• 24.Perform LC-MS experiment with an Easy-nLC 1200 chromatography system with a 50 cm × 75 μm inner diameter Easy-Spray reverse phase column (C-18, 2 μm particles) coupled to an Orbitrap Fusion Lumos mass spectrometer. Analytical column equilibration was performed before every sample run.

Note: Prior to running cross-linked samples, the mass spectrometer should be calibrated, and a quality control sample should be performed to ensure LC-MS performance for LC chromatographic response and MS instrument response.

• 25.Load 2 μL of peptides for LC-MS analysis. 3 technical replicates to be acquired per biological replicate. LC gradient is listed in Table 1.

Note: We recommend that 3 cross-linking (technical) replicates are sufficient per biological replicate. Due to the stochastic nature of mass spectrometry fragmentation and sequencing, the technical replicates help to improve the chances of identifying cross-linked peptides by data-dependent acquisition, which can be further validated using targeted acquisition. We recommend that a minimum of 2 biological replicates be performed.

Note: Gradient can be adjusted to increase or decrease separation time. Table 1 shows an example 60 min separation time (1–61 min) from 3-42% solvent B. A 90 min separation time can be used to increase time for peptide acquisition, and the percentage of solvent B used can be adjusted to 3–35% solvent B to capture more hydrophilic peptides.

• 26.For DSSO cross-linked peptides, DDA method using MS2-MS3 fragmentation to be used for MS acquisition.

Note: MS2 performed for ions with charges between 3 to 8. MS3 performed on ions with mass difference of 31.9721 (signature fragmentation of MS-cleavable DSSO cross-link). Full details of MS parameters are listed in Table 2.

• 27.For SDA cross-linked peptides, DDA method using MS2 fragmentation to be used for MS acquisition.

Note: MS2 performed for ions with charges between 3 and 8. Full details of MS parameters are listed in Table 2.

• 28.
  MS raw files were searched using MetaMorpheus.⁴ (Figure 1A; Table 3)

  • a.
    Load the fasta file with protein sequence of antibody and HLA into MetaMorpheus.
  • b.
    Load the MS .raw files into MetaMorpheus.
  • c.
    Add calibration task.
  • d.
    For DSSO cross-links, add XL search task for DSSO. (Figure 1B).
  • e.
    For SDA cross-links, add 2 XL search task, SDA (amine-to-any) and SDA (amine-to-acid).
• 29.From MetaMorpheus output folder, open the Interlinks.tsv output file.
• 30.
  Filter Interlinks based on following criteria.

  • a.
    Q-value ≤ 0.01 (corresponds to a 1% FDR rate).
  • b.
    Cross-links are between antibody light or heavy chain to HLA alpha-chain.
  • c.
    Cross-link site on antibody light or heavy chain falls on a residue that is part of or near (within 10 amino acids) of the Complementary Determining Region of the antibody.
• 31.From the filtered cross-link list from step 29, make a list of the Precursor MZ and Precursor Charge values, which will be used for targeted XL-MS.

Table 1.

Gradient for liquid chromatography for LC-MS

Time (min)	Flow rate (nL/min)	Percentage of Solvent A (0.1% formic acid in water)	Percentage of Solvent B (95% ACN, 0.1% formic acid in water)
0	300	97	3
1	300	97	3
61	300	58	42
66	300	50	50
71	300	10	90
75	300	10	90

Table 2.

MS parameters for discovery cross-linking mass spectrometry acquisition

MS parameters	MS2-MS3 (DSSO cross-linker)	MS2 only (SDA cross-linker)
MS1 parameters
Scan range	350–1600 m/z	350–1600 m/z
Mass analyzer	Orbitrap	Orbitrap
Mass resolution	60,000	60,000
AGC target	400,000	400,000
Max. inject time	50 ms	50 ms
Selection for MS2 parameters
Charge state	3 to 8	3 to 8
Dynamic exclusion	60 s	60 s
Minimum Intensity Threshold	20,000	20,000
MS2 parameters
Isolation window	1.6 m/z	1.6 m/z
Collision mode	CID	HCD
Collision energy	30%	30%
Mass analyzer	Orbitrap	Orbitrap
Mass resolution	30,000	30,000
AGC target	50,000	50,000
Max. inject time	70 ms	54 ms
Selection for MS3 parameters
Targeted mass difference	31.9721 m/z	-
MS3 parameters
Collision mode	HCD	-
Collision energy	30%	-
Mass analyzer	Orbitrap	-
Mass resolution	30,000	-
AGC target	50,000	-
Max. inject time	100 ms	-

Figure 1.

Setting up search of discovery cross-linking mass spectrometry data

(A) User interface for setting up MetaMorpheus search which requires protein sequence fasta file, mass spectrometry raw files and task settings files.

(B) Example of parameters used in task setting file for DSSO cross-link search.

Table 3.

Search parameters on MetaMorpheus for discovery cross-linking mass spectrometry data

Search parameters	DSSO task	SDA (amine-to-any) task	SDA (amine-to-acid) task
CrosslinkerModSites	KSTY	KSTY	KSTY
CrosslinkerModSites2	KSTY	X	DE
Quench Methods H2O	Checked	Checked	Checked
Quench Methods Tris	Checked	Checked	Checked
MS2 Dissociation Type	CID	HCD	HCD
MS2 Child Scan Dissociation	Null	Null	Null
MS3 Child Scan Dissociation	HCD	Null	Null
Crosslink at Cleavage Site	Checked	Checked	Checked
MaxMissedCleavages	3	3	3
Protease	trypsin	Trypsin	Trypsin
Precursor Mass Parameters	10 ppm	10 ppm	10 ppm
Product Mass Tolerance	20 ppm	20 ppm	20 ppm
Static modifications	Carbamidomethylation, +57.0215 (C)	Carbamidomethylation (C)	Carbamidomethylation (C)
Variable modifications	Oxidation, +15.9949 (M)	Oxidation, +15.9949 (M)	Oxidation, +15.9949 (M)
	DSSO_alkene, +54.0106 (KSTY)	SDA_alkene, +68.0262 (KSTY)	SDA_alkene, +68.0262 (KSTY)
	DSSO_hydro, +176.0143 (KSTY)	SDA_hydro, +100.0524 (KSTY)	SDA_hydro, +100.0524 (KSTY)
	DSSO_tris, +279.0777 (KSTY)	SDA_Tris, +203.1158 (KSTY)	SDA_Tris, +203.1158 (KSTY)
	DSSO_loop, +158.0038 (KSTY)	SDA_loop, +82.0419 (KSTY)	SDA_loop, +82.0419 (KSTY)
	DSSO_thiol, +85.9826 (KSTY)	SDA_N2, +110.0480 (KSTY)	SDA_N2, +110.0480 (KSTY)
		SDA_oxid, +98.0368 (KSTY)	SDA_oxid, +98.0368 (KSTY)

Targeted cross-linking mass spectrometry and data analysis to validate cross-link pairs between antibody to HLA

Timing: 1–2 days

Targeted cross-linking mass spectrometry performed for cross-linked peptides between antibody to HLA.

• 32.Prepare the targeted MS acquisition method (Figure 2A) by modifying the MS acquisition method in steps 25–26 by adding an additional experiment to the MS method with the parameters in Table 4.
• 33.Input the Mass List Table based on the list of the Precursor MZ and Precursor Charge values from step 30.
• 34.Load 2 μL of peptides for LC-MS analysis. 3 technical replicates to be acquired per biological replicate.
• 35.Perform LC-MS experiment with same LC-MS system as described in step 23 with the prepared targeted-XL-MS method in steps 31–32.
• 36.After MS acquisition, prepare a .txt input file for Cross-link Transition Calculator,⁶ which requires the short and long mass of cross-linker (if cleavable) and peptideA, peptideB sequence and masses and charge state of precursor ion. (Figure 2B).
• 37.Load acquired targeted MS .raw files into Skyline⁵ with Cross-link Transition Calculator plugin with the loaded cross-linked peptide information from step 35.

Note: Skyline should load and extract MS peaks based on the cross-linked peptide information provided.

• 38.Inspect cross-link peptide peaks, noting their mass accuracy, peak shape and reproducibility of identification across at least three replicates.

Note: Cross-linked peptides that are reproducibility identified by targeted XL-MS can be used for structural modeling of the antibody-HLA interaction.

Figure 2.

Setting up targeted cross-linking mass spectrometry acquisition

(A) MS method editor parameters, including experiment for targeted MS2 based on cross-linked peptide mass and charge in mass list table.

(B) Format of cross-linked peptides to input for cross-link transition calculator in skyline.

Table 4.

MS parameters for targeted cross-linking mass spectrometry acquisition

MS parameters	Targeted XL-MS
MS level	2
Isolation window	1.6 m/z
Activation type	HCD
HCD collision energy	32%
Mass analyzer	Orbitrap
Mass resolution	15,000
Max. inject time	80 ms

Structural modeling of antibody-HLA interaction using cross-linking data

Timing: 1 week

After identifying high confidence cross-linked peptides for antibody to HLA interaction using discovery and targeted XL-MS, these cross-linked peptides can be used to model the interaction structure using predicted structures and molecular docking.

• 39.
  To perform molecular docking using HADDOCK,⁹ the following structures and information are needed:

  • a.
    Structure of the antibody protein in PDB file format.
  • b.
    Structure of the HLA protein in PDB file format.
  • c.
    Amino acids on antibody that are predicted to interact with HLA.
  • d.
    Amino acids on HLA that are predicted to interact with antibody (predicted eplets).
  • e.
    Cross-link sites between antibody to HLA prepared as a HADDOCK restraints TBL file.

Note: Antibody structure can be predicted using ABodyBuilder.^7,10 Antibody paratope sites can be predicted using Antibody i-Patch.⁸ Predicted eplets on HLA can be obtained using HLA Matchmaker with Luminex assay data.¹¹

• 40.Load HLA protein structure and antibody structure onto HADDOCK web server for submission.
• 41.Load amino acids that are predicted to interact as active residues of antibody or of HLA under Input parameters tab. (Figure 3A).
• 42.Load cross-link restraints file under Docking parameters tab. (Figures 3B and 3C).
• 43.Submit the structural and restraint inputs on HADDOCK.
• 44.After job has completed, download the result files and docked structure of antibody to HLA interaction guided by XL-MS data. Structures can be viewed in PyMOL.

Figure 3.

Input for cross-linking mass spectrometry data and interacting surface data for molecular docking using HADDOCK

(A) List interacting amino acids for antibody and HLA proteins under active residues as part of input parameters.

(B) Load cross-link sites as a HADDOCK restraints TBL file under Distance restraints for Docking parameters.

(C) Example HADDOCK restraints file format for cross-links.

Expected outcomes

The current protocol was performed on three different antibodies that bind to HLA-A∗11:01.¹ After cross-linking, a representative stained protein gel should indicate that antibody is cross-linked to HLA (Figure 4A). After performing cross-linking and discovery XL-MS, 4 inter-links between Ab to HLA were identified (Figure 4B). The 4 inter-links were then further investigated using targeted XL-MS, with two of the four inter-links identified reproducibly by targeted XL-MS. An example of the targeted XL-MS identification of one of the inter-links is shown in Figure 4C. Using the cross-links for molecular docking of Ab and HLA structures produced different structural models (Figures 4E–4G) depending on whether cross-link data was used as restraint and whether predicted interacting amino acids were used as restraint. Crystal structure of the Ab to HLA interaction is shown as a reference (Figure 4D). We recommend using both cross-links identified from both discovery and targeted XL-MS together with predicted interacting amino acids to achieve a model closest to the crystal structure.

Limitations

Current protocol requires a molecular characterization of antibody and HLA protein pair. Antibody sequence and HLA sequence are required, in addition to being able to express and purify both antibody and HLA protein to perform cross-linking mass spectrometry.

Another limitation is that the number of identified cross-link pairs between antibody to HLA protein is low, with expected less than 10 cross-links identified after both discovery-based and targeted-based XL-MS. The low number of cross-links may affect accuracy of molecular docking to predict the interacting structure of antibody to HLA pair. The antibody-HLA pair that was characterized by Ser et al.¹ demonstrated a 4-6 Å difference between the molecular docking model and the crystal structure for hydrogen bonding residues. However, this discrepancy is likely to vary depending on antibody-HLA pair used, quality of cross-linking data and predicted inputs. Care must be taken in using the predicted structural model, as kinetics and dynamics of binding are not captured by the predicted model.

Troubleshooting

Problem 1

Antibody was not cross-linked to HLA protein.

Potential solution

Several possible causes may contribute to lack of cross-linking of antibody to HLA protein, pertaining to steps 1–13.

• •Check that antibody interacts with HLA protein using an ELISA assay to test binding. This will eliminate possibility that proteins are degraded.
• •Test antibody to HLA molar ratios for cross-linking. Example antibody to HLA molar ratios to test are 1:1 and 1:2.
• •Certify that purified proteins are in amine-free buffer. Buffer exchange to remove amine-based buffers.
• •Check that cross-linking of proteins occurred by running an SDS-PAGE gel and staining with Coomassie. Cross-linked proteins should show a shift in molecular weight when compared to non-cross-linked protein.
• •If no cross-linked protein bands are observed in SDS-PAGE gel, optimize cross-linking conditions by increasing concentration of cross-linker and incubation times (or UV activation times) to increase cross-linking reaction.
• •Test alternative cross-linkers, such as disuccinimidyl glutarate (DSG) or N,N′-carbonyldiimidazole (CDI), which have shorter spacer arms.

Problem 2

No cross-links identified between antibody to HLA by discovery cross-linking mass spectrometry.

Potential solution

We recommend to ensure that proteins are cross-linked before proceeding to mass spectrometry acquisition (see problem 1, steps 1–13). After confirming that proteins are cross-linked, but cross-links are still not being identified by discovery cross-linking mass spectrometry, we recommend to check the following (steps 14–25):

• •Increase amount of cross-linked proteins used for MS acquisition. 20–50 μg of starting protein for cross-linking should be sufficient amount for downstream MS acquisition, with 1 μg of digested peptides loaded on column should be sufficient for detection.
• •Increase LC separation time to 90 min (see Table 1).
• •Check that MS parameters are set correctly for acquisition of MS data.

Problem 3

Identified cross-links between antibody to HLA do not fall on complementary determining regions (CDR) of antibody, or do not seem to fall on interacting regions of antibody to HLA.

Potential solution

Cross-links are identified for antibody and for HLA, but not to the desired interacting region between antibody to HLA. We recommend checking the following:

• •Check that antibody interacts with HLA protein using an ELISA assay to test binding. This will eliminate possibility that proteins are degraded.
• •Test alternative cross-linkers, such as disuccinimidyl glutarate (DSG) or N,N′-carbonyldiimidazole (CDI), which have shorter spacer arms, or such as LC-SDA, which has longer spacer arm. The different spacer arm may provide cross-linking at the interacting surface.
• •Check protein sequences of antibody and HLA protein to determine if there are lysines available near the CDR regions for cross-linkers to react with. If lysines are not present, the antibodies may not be suitable for cross-linking mass spectrometry using amine-reactive cross-linkers.

Problem 4

Targeted cross-linking mass spectrometry does not identify cross-linked peptides from list obtained by discovery cross-linking mass spectrometry.

Potential solution

Check that the mass of the cross-linked peptides used for the mass list table for targeted acquisition are accurate, and that they account for protonated mass based on charge state. Check that the charge state listed is accurate. These checks can be done by cross-referencing against the acquired discovery cross-linking mass spectrometry data. After the mass and charge of the targeted cross-linked peptides have been confirmed, and the cross-linked peptides is still not observed by targeted XL-MS, these peptides are likely low confidence identified cross-linked peptides.

Problem 5

Structure of antibody to HLA based on molecular docking is not accurate.

Potential solution

Accuracy of molecular docking interaction structure of antibody to HLA depends on the accuracy of inputs used for molecular docking. Lower confidence cross-links or low confidence predicted interacting amino acids will need to be removed as molecular docking constraints to improve accuracy of the resultant structural model.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Radoslaw Sobota (rmsobota@imcb.a-star.edu.sg).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to the technical contact, Zheng Ser (ser_zheng@imcb.a-star.edu.sg).

Materials availability

This study did not generate specialized materials.

Data and code availability

This study did not generate datasets or code.