Protocol for identification of protein citrullination by immunoprecipitation followed by mass spectrometry

Summary

Protein citrullination occurs through the deimination of peptidyl-arginine to yield peptidyl-citrulline by one of the peptidyl-arginine deiminase (PAD) family members. This protocol identifies citrullinated protein residues using immunoprecipitation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We describe steps for identification of citrullination modifications in vitro or in cell culture, immunoprecipitation of nuclear citrullinated proteins, and identification of citrullinated residues by mass spectrometry (MS). This protocol applies to both recombinant protein assays and in vitro cell culture assays.

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

Graphical abstract

Highlights

• •Practical guidance for enriching citrullinated proteins
• •Optimized steps for isolating and immunoprecipitating large-scale nuclear extracts
• •Mass spectrometry-based workflow for confident identification of citrullination sites

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

Protein citrullination occurs through the deimination of peptidyl-arginine to yield peptidyl-citrulline by one of the peptidyl-arginine deiminase (PAD) family members. This protocol identifies citrullinated protein residues using immunoprecipitation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We describe steps for identification of citrullination modifications in vitro or in cell culture, immunoprecipitation of nuclear citrullinated proteins, and identification of citrullinated residues by mass spectrometry (MS). This protocol applies to both recombinant protein assays and in vitro cell culture assays.

Before you begin

Citrullination is a post-translational modification that results in the conversion of arginine within proteins to the non-natural amino acid citrulline.² First described at length in neutrophil activation,³ citrullination is now appreciated as an important post-translational modification that regulates protein activity, localization, stability, and the ability to bind to chromatin or other protein factors.^4,5,6,7 Citrullination is carried out by four of the five PAD family members: PADI1, PADI2, PADI3, PADI4. PADI6 is believed to be catalytically inactive. While the repertoire of known citrullinated proteins is still limited, a growing list of proteins are being identified as targets of citrullination including histones, HIF1α, and p53.^1,8

It is recommended that researchers use a system with increased citrullination activity to allow for sufficient capture of citrullinated residues. While some cells possess intrinsically high PAD activity (i.e., neutrophils express high levels of PADI4), other cell types (such as certain cancers) may possess low, but physiologically meaningful, PAD activity.⁹ It is recommended to enhance citrullination either through PAD enzyme activation through treatment with calcium ionophore, or through upregulation of PAD enzymes themselves. Citrulline residues that are identified can then be validated under more physiological conditions. Additionally, we recommend establishing a robust positive control system using in vitro citrullination reactions, which are described for PADI4 enzymatic targets in this protocol.

Due to the high cell number requirement for this method and costs associated with antibodies and mass spectrometry, we advise researchers to perform a pilot experiment to optimize the amount of protein and antibody required for efficient immunoprecipitation. Please see our suggested optimization guidelines as outlined in the troubleshooting subsection to ensure high quality results.

The protocol below is applicable for in vitro assays using recombinant proteins or nuclear protein complexes isolated from cells, as well as a variety of cultured immortalized cell lines. However, optimization may be required for recombinant proteins isolated from different expression systems. The following protocol was successfully performed on in vitro recombinant p53 isolated from baculovirus and p53 immunoprecipitated from HCT116 colon cancer cells with tetracycline-inducible PADI4.

• 1.Prepare samples using the in vitro citrullination assay for a positive control.
• 2.Grow ten 15 cm plates of HCT116 cells per condition (10 x 15 cm plates = approximately 6 x 10⁷ cells). Cells should be about 70-80% confluent.
• 3.Prepare all buffers as described. Prepare enough of each buffer for all conditions. Filter-sterilize buffers as indicated with 0.22 μm filters.
• 4.Add protease inhibitors to the buffers where indicated.

CRITICAL: Because mass spectrometry is highly sensitive, preventing personal and airborne sample contamination, especially with keratin, is essential. To minimize the risk of contamination, wear a clean lab coat throughout all procedures, thoroughly clean the bench area used for sample preparation before starting, change gloves frequently, and avoid leaving samples, solutions, or reagents exposed for extended periods. See the troubleshooting 3 for more information.

Innovation

This protocol describes the identification of citrullinated residues from both purified proteins and immunoprecipitated targets of interest. Citrullination has historically been a difficult modification to identify due to the low cellular abundance and the small change in mass (+0.9840 Da) that is indistinguishable from the more common deamidation of asparagine and glutamine. The innovation of this method lies in the integration of citrullinated protein enrichment with an optimized proteomics workflow that enables comprehensive identification of citrullinated sites. The enrichment step enhances sequence coverage of the protein of interest, and the proteomics strategy that incorporates multiple protease digestions, high-resolution MS analysis, refined database search parameters, and detection of the diagnostic −43.0058 Da neutral loss of isocyanic acid (HNCO) during peptide fragmentation,^10,11 enables unambiguous identification of citrullination residues. These findings can be further validated by additional downstream methods in tissues and cell culture. We have confirmed the presence of citrulline residues identified by this method in mouse tissue and in multiple cancer cell lines. Together, these advances provide a practical and reproducible workflow for the identification of citrulline residues on a protein of interest, and will enable the scientific community to expand its research on the role of citrullination in biology and disease.

Institutional permissions

Researchers should obtain permission from the relevant institutions before conducting any BSL-2 level research with recombinant DNA constructs and mammalian cell lines.

In vitro citrullination reaction

Timing: 2 h

The purpose of this step is to perform in vitro citrullination of the target protein. PAD enzymes do not use a cofactor, but calcium ions should be in molar excess to the enzyme to ensure maximal activity of the PAD enzyme. Prior to beginning this step, the PAD enzyme of interest and target protein of interest should be purified to > 95% purity or purchased from companies with demonstrated enzymatic activity and stored in an EDTA-free buffer, as EDTA can inactivate PAD enzymes.

• 5.Calculate the concentration of PADI4 or PAD enzyme of interest to add to the citrullination reaction mixture, using a molar ratio of approximately 1:100 of PAD enzyme to target protein.
• 6.
  In a 1.5 mL microfuge tube, add the following reagents:

  • a.
    PAD Enzyme Buffer
  • b.
    Protein target of interest
  • c.
    PAD enzyme

Note: For PADI4 reactions, it is recommended to use recombinant mononucleosomes + PADI4 as a positive control, and recombinant mononucleosomes + PADI4 + the PADI4 inhibitor 50-500 nM GSK484 as a negative control. It is also recommended to incubate your target protein + PADI4 with the PADI4 inhibitor GSK484 as a negative control.

• 7.Mix thoroughly and incubate at 37°C in a ThermoMixer or heat block for 1 h with gentle agitation (400 rpm).
• 8.Immediately inhibit the reaction by adding 4X SDS-PAGE Loading Buffer and boil samples for 10 min at 95°C to stop the citrullination reaction.
• 9.Determine the reaction efficiency using 4-20% SDS-PAGE electrophoresis gel and an antibody targeting citrulline residues, such as mouse polyclonal peptidyl-citrulline (clone F95).

Note: If using recombinant mononucleosomes as a positive control, the antibody targeting H3 citrulline sites (citrulline R2 + R8 + R17) may also be used. See Indeglia et al.¹ for an example using this system.

Cell culture and activation of PAD enzyme

Timing: 2–5 days

• 10.Culture and maintain an inducible cell line, such as HCT116 cells with tetracycline inducible PADI4 and tetracycline inducible empty vector, at 37°C and 5% CO₂ in 15 cm culture plates in McCoy’s 5A medium supplemented with 10% tetracycline-free FBS and 1% penicillin/streptomycin.

Note: If your experiment requires cell treatments, please account for the additional steps and processing time involved. Refer to the treatments described in Indeglia et al., 2025, which used doxycycline to activate the tetracycline-inducible PADI4 and nutlin-3a to activate p53.

• 11.Add fresh media with the appropriate drug treatments: 50 ng/mL doxycycline and 10 μM nutlin-3a.
• 12.Incubate cells for an additional 24 h.

Note: It is also recommended at this step to confirm citrullination activity in cell cultures by SDS-PAGE gel and an antibody targeting citrulline residues. The presence of a citrullinated band at approximately the molecular weight of the protein of interest will greatly increase the chances of a successful downstream MS analysis. See Indeglia et al.¹ for more information on confirming citrullination activity by SDS-PAGE.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse monoclonal anti-p53 (DO1) (human specific) (recommended starting dilution for IP, 2 μg per 1 x 107 cells. For WB, 1:1000)	Santa Cruz Biotechnology	Cat# sc-126; RRID:AB_628082
Mouse monoclonal anti-Histone H3 (1B1B2) (recommended starting dilution for WB, 1:1000)	Cell Signaling Technology	Cat# 14269; RRID:AB_2756816
Mouse monoclonal IgG1 (G3A1) Isotype Control (dilution is dependent on target of interest)	Cell Signaling Technology	Cat# 5415S; RRID:AB_10829607
Rabbit polyclonal anti-PADI4 (recommended starting dilution for IP, 10 μg per 1 x 107 cells. For WB, 1:1000)	ProteinTech	Cat# 17373-1-AP; RRID:AB_2878398
Mouse polyclonal peptidyl-citrulline (clone F95) (recommended starting dilution for WB, 1:500)	MilliporeSigma	Cat# MABN328; RRID:AB_2938608
Rabbit polyclonal anti-Histone H3 (citrulline R2 + R8 + R17) (recommended starting dilution for WB, 1:1000)	Abcam	Cat# ab5103; RRID:AB_304752
Chemicals, peptides, and recombinant proteins
0.5M EDTA	Thermo Fisher Scientific	Cat# 15575-038
20% SDS Solution	Bio-Rad	Cat# 1610418
Acetonitrile, Optima LC/MS Grade	Thermo Fisher Scientific	Cat# A955-4
Ammonium bicarbonate	Sigma-Aldrich	Cat# A6141
Beta-mercaptoethanol (BME)	Thermo Fisher Scientific	Cat # 21985023
BS3 (bis(sulfosuccinimidyl)suberate)	Thermo Fisher Scientific	Cat # 21580
CaCl2	Sigma-Aldrich	Cat# 21115
Chymotrypsin, Sequencing Grade	Promega	Cat# V1061
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail	Sigma-Aldrich	Cat# 11836170001
DMSO (Dimethyl Sulfoxide)	Cell Signaling Technology	Cat# 12611P
Doxycycline-HCl	Thermo Fisher Scientific	Cat# J60422-14
DPBS (Dulbecco’s Phosphate-Buffered Saline)	Corning	Cat# 21-031-CV
DTT (dithiothreitol)	Thermo Fisher Scientific	Cat# R0861
Dynabeads Protein G for Immunoprecipitation	Thermo Fisher Scientific	Cat# 10004D
Formic acid, Optima LC/MS Grade	Thermo Fisher Scientific	Cat# A117
Glycerol	Thermo Fisher Scientific	Cat# 410980100
GSK484	MedChem Express	Cat# HY-100514
HEPES	Corning	Cat# 25-060-CI
IgG Elution Buffer	Thermo Fisher Scientific	Cat# 21004
Immobilon -P PVDF 0.45μm Membrane	Sigma-Aldrich	Cat# IPVH00010
Iodoacetamide	Sigma-Aldrich	Cat# I1149
KCl	Thermo Fisher Scientific	Cat# 410980100
Laemmli SDS sample buffer, reducing (6X)	Thermo Fisher Scientific	Cat# J61337.AD
LightShift Poly (dI-dC)	Thermo Fisher Scientific	Cat# 20148E
McCoy’s 5A (Iwaketa and Grace Modification)	Corning	Cat# 10-050-CV
Methanol	Thermo Fisher Scientific	Cat# BP1105-4
Methanol, Optima LC/MS Grade	Thermo Fisher Scientific	Cat# A456-4
MgCl2	Thermo Fisher Scientific	Cat# AM9530G
NaCl	Thermo Fisher Scientific	Cat# AM97060
NP-40 (10%)	Thermo Fisher Scientific	Cat# 85124
NuPAGE Mini Protein Gels (10% Tris-Glycine, 4-20% Tris-Glycine)	Thermo Fisher Scientific	Cat# XP00100
NuPAGE MOPS SDS	Thermo Fisher Scientific	Cat# NP0001
Nutlin-3a	Sigma-Aldrich	Cat# SML0580
PageRuler Prestained Protein Ladder	Thermo Fisher Scientific	Cat# 26616
Penicillin/Streptomycin	Thermo Fisher Scientific	Cat# 15140122
Phenylmethylsulfonyl fluoride (PMSF)	Thermo Fisher Scientific	Cat# 36978
PhosSTOP phosphatase Inhibitor	Sigma-Aldrich	Cat# 04906837001
Recombinant mononucleosomes	Active Motif	Cat# 81071
Recombinant p53	Active Motif	Cat# 81091
Recombinant PADI4	Cayman Chemicals	Cat# 25915
Sequencing Grade Modified Trypsin	Promega	Cat# V5111
Sodium phosphate	Thermo Fisher Scientific	Cat# 447982500
Trifluoroacetic acid (TFA), Optima LC/MS Grade	Thermo Fisher Scientific	Cat# A116
Tris-(2-Carboxyethyl)phosphine, Hydrochloride (TCEP)	Thermo Fisher Scientific	Cat# PG82089
Tris Buffered Saline with Tween 20	Cell Signaling Technology	Cat# 9997
Tris-HCl, pH 7.5	Thermo Fisher Scientific	Cat# 15567027
Tris-HCl, pH 8.0	Thermo Fisher Scientific	Cat# 15568025
Tris-HCl, pH 9.5	Thermo Fisher Scientific	Cat# J62084.K2
Trypan Blue Solution	Corning	Cat# 25-900-CI
Trypsin-EDTA (0.05%)	Thermo Fisher Scientific	Cat# 25300-054
Critical commercial assays
BCA Protein Assay	Bio-Rad	Cat# 5000001
Colloidal Blue Staining Kit (or similar)	Thermo Fisher Scientific	Cat# LC6025
Pierce ECL Western Blotting Substrate	Thermo Fisher Scientific	Cat# 32106
Slide-A-Lyzer Accessory Float Buoys and Syringes	Thermo Fisher Scientific	Cat# 66430
Slide-A-Lyzer Dialysis Cassettes	Thermo Fisher Scientific	Cat# 66383
Experimental models: Cell lines
HCT116 tet-PADI4	Indeglia et al.1	N/A
Software and algorithms
MaxQuant 2.2.0.0 or later	Cox and Mann12	RRID:SCR_014485
pFind 3.2.1	Chi et al.13	RRID:SCR_003011
Other
0.5 mL Protein LoBind microfuge tubes	Eppendorf	Cat# 13-698-793
Acclaim PepMap 100 C18 Trap column	Thermo Fisher Scientific	Cat# 164535
Autosampler Vials (QuanRecovery)	Waters	Cat# 186009186
Caps for Autosampler Vials	Waters	Cat# 186000274
Captair Bio PCR workstation	erlab	Cat# RNA/DNA-110-2011-CN
CentriVap Vacuum Concentrator	Labconco	Cat# 7810010
Constant temperature incubator shaker	Taitec	Cat# MBR-024
Countess and Cell Counting Chamber Slides	Thermo Fisher Scientific	Cat# C10228
Dounce homogenizer	Thermo Fisher Scientific	Cat# 501945204
DynaMag-15 Magnet	Thermo Fisher Scientific	Cat# 12301D
DynaMag-2 Magnet	Thermo Fisher Scientific	Cat# 12321D
Labnet International, Inc H5600 REVOLVER ROTATOR	Thermo Fisher Scientific	Cat# NC9909880
Milli-Q Ultrapure Water Purification System	Millipore	Cat# ZIQ7000TOC
nanoEase M/Z Peptide BEH C18 column	Waters	Cat# 186008795
Q Exactive Plus mass spectrometer	Thermo Fisher Scientific	N/A
Razor blades	AccuTec	Cat# 94-0474-0000
Shaking thermomixer	Thermo Fisher Scientific	Cat# 88880028
Vanquish Neo UHPLC system	Thermo Fisher Scientific	Cat# VN-S10-A-01

Materials and equipment

PAD Enzyme Buffer

Reagent	Final concentration	Amount
1 M HEPES	50 mM	500 μL
1 M CaCl2	10 mM	100 μL
5 M NaCl	50 mM	100 μL
1 M DTT	5 mM	50 μL
ddH2O	N/A	9.25 mL
Total	N/A	10 mL

Note: Store at 4°C for up to 1 month. Add DTT immediately before use.

Swelling Buffer

Reagent	Final concentration	Amount
1 M HEPES	10 mM	1 mL
1 M MgCl2	1.5 mM	150 μL
2 M KCl	10 mM	500 μL
1 M DTT	0.5 mM	50 μL
ddH2O	N/A	97.8 mL
Total	N/A	100 mL

Note: Store at 4°C for up to 1 month. Add DTT, 1 mg/mL each of protease inhibitors aprotinin, leupeptin, and pepstatin immediately before use.

Nuclear Lysis Buffer

Reagent	Final concentration	Amount
1 M HEPES	20 mM	2 mL
1 M MgCl2	1.5 mM	150 μL
100% w/v Glycerol	25% w/v	25 mL
0.5 M EDTA	0.2 mM	40 μL
5 M NaCl	0.42 M	8.4 mL
1 M DTT	0.5 mM	50 μL
100 mM Phenylmethylsulfonyl fluoride (PMSF)	0.2 mM	200 μL
ddH2O	N/A	64.16 mL
Total	N/A	100 mL

Note: Store at 4°C for up to 1 month. Add DTT, 1 mg/mL each of protease inhibitors aprotinin, leupeptin, and pepstatin immediately before use.

Dialysis Buffer

Reagent	Final concentration	Amount
1 M Tris HCl, pH 8.0	20 mM	80 mL
2 M KCl	80 mM	160 mL
100% w/v Glycerol	10% w/v	400 mL
0.5 M EDTA	0.2 mM	1.6 mL
Beta-mercaptoethanol (BME)	1 mM	1.8 mL
100 mM Phenylmethylsulfonyl fluoride (PMSF)	0.2 mM	8 mL
ddH2O	N/A	3.3486 L
Total	N/A	4 L

Note: Store at 4°C for up to 1 week. Add BME and PMSF immediately before use.

BS³ Conjugation Buffer

Reagent	Final concentration	Amount
1 M Sodium phosphate	20 mM	200 μL
5 M NaCl	0.15 M	300 μL
ddH2O	N/A	9.5 mL
Total	N/A	10 mL

BS³ Quenching Buffer

Reagent	Final concentration	Amount
1 M Tris HCl, pH 7.5	N/A	1 mL
Total	N/A	1 mL

Immunoprecipitation (IP) Buffer

Reagent	Final concentration	Amount
1 M Tris, pH 8.0	20 mM	2 mL
5 M NaCl	100 mM	2 mL
10% NP-40	0.1%	1 mL
ddH2O	N/A	95 mL
Total	N/A	100 mL

Note: Store at 4°C for up to 1 month. Add 1 mg/mL each of protease inhibitors aprotinin, leupeptin, and pepstatin immediately before use.

Reducing Solution

Reagent	Final concentration	Amount
Ammonium bicarbonate	25 mM	0.198 g
Tris-(2-carboxyethyl) phosphine (TCEP)	20 mM	0.573 g
Milli-Q water	N/A	100 mL
Total	N/A	100 mL

Note: Adjust pH to 8.0 with 5 M NaOH. Store aliquots at −20°C for up to 6 months.

Alkylating Solution

Reagent	Final concentration	Amount
Ammonium bicarbonate	25 mM	0.198 g
Iodoacetamide	25 mM	0.463 g
Milli-Q water	N/A	100 mL
Total	N/A	100 mL

Note: Iodoacetamide is light sensitive. Store aliquots in dark at −20°C for up to 6 months.

• •0.4 M ammonium bicarbonate, pH 8.0

Dissolve 15.81 g ammonium bicarbonate in 500 mL Milli-Q water. Filter solution using a 0.22 μm filter.

Note: The pH should be 8 without adjustment. Store at 4°C for up to 3 months.

• •50 mM ammonium bicarbonate, pH 8.0

Add 1.25 mL 0.4 M ammonium bicarbonate to 8.75 mL MilliQ water.

Note: Prepare fresh before use. Store at 20-25°C.

• •Destaining Solution

Add 10 mL 0.4 M ammonium bicarbonate to 10 mL LC-MS grade acetonitrile. Final concentration is 0.2 M ammonium bicarbonate, 50% acetonitrile.

Note: Prepare fresh before use. Store at 20-25°C.

• •Wash Buffer 1

Combine 5 mL of 50 mM ammonium bicarbonate with 5 mL LC-MS grade acetonitrile. Final concentration is 25 mM ammonium bicarbonate, 50% acetonitrile.

Note: Prepare fresh before use. Store at 20-25°C.

• •Wash Buffer 2

Combine 5 mL of 50 mM ammonium bicarbonate with 5 mL Milli-Q water. Final concentration is 25 mM ammonium bicarbonate.

Note: Prepare fresh before use. Store at 20-25°C.

• •Trypsin Stock Solution

Dissolve one vial of 20 μg lyophilized sequencing-grade modified trypsin in 40 μl trypsin resuspension buffer provided by the manufacturer. Final concentration is 0.5 μg/μl.

Note: Aliquot and store at −20°C for up to 3 months.

• •Protease Digest Solution

Add 10 μl Trypsin Stock Solution to 215 μl Milli-Q water and 25 μl 0.4 M ammonium bicarbonate. Final concentration is 20 ng/μl trypsin, 40 mM ammonium bicarbonate.

Note: Prepare on ice immediately before use.

• •Digest Dilution Buffer

Add 500 μl 0.4 M ammonium bicarbonate to 4.5 mL Milli-Q water. Final concentration is 40 mM ammonium bicarbonate.

Note: Prepare fresh before use. Store at 20-25°C.

• •Extract Solution 1

Add 5 μl LC-MS grade formic acid to 5 mL Milli-Q water. Final concentration is 0.1% (v/v) formic acid.

Note: Store at 20-25°C for up to 1 week.

• •Extract Solution 2

Add 5 μl LC-MS grade formic acid to 1 mL Milli-Q water and 4 mL of LC-MS grade acetonitrile. Final concentration is 0.1% (v/v) formic acid, 80% acetonitrile.

Note: Store at 20-25°C for up to 1 week.

• •0.1% (v/v) TFA

Add 5 μl LC-MS grade trifluoroacetic acid to 5 mL Milli-Q water. Final concentration is 0.1% (v/v) trifluoroacetic acid.

Note: Store at 20-25°C for up to 1 week.

• •HPLC Solvent A

Add 500 μl LC-MS grade formic acid to 499.5 mL Milli-Q water. Final concentration is 0.1% (v/v) formic acid.

Note: Keep at 20-25°C for up to 1 month.

• •HPLC Solvent B

Add 500 μl LC-MS grade formic acid to 499.5 mL LC-MS grade acetonitrile. Final concentration is 0.1% (v/v) formic acid in acetonitrile.

Note: Keep at 20-25°C for up to 1 month.

CRITICAL: Acetonitrile is flammable. Store in a certified flammable safety cabinet. Acetonitrile can cause eye irritation and is harmful if inhaled, absorbed through the skin, or ingested. Always handle in a fume hood. Wear appropriate personal protective equipment (PPE), including chemical-resistant gloves, lab coat or protective clothing, and safety goggles.

CRITICAL: Methanol is flammable. Store in a certified flammable safety cabinet. Methanol is toxic if swallowed. May cause eye damage, skin irritation, and respiratory issues if inhaled. Use only in a fume hood. Wear appropriate PPE, including chemical-resistant gloves, lab coat or protective clothing, and safety goggles.

CRITICAL: Always handle concentrated formic and trifluoroacetic acids in a certified chemical fume hood to prevent inhalation of harmful vapors. Acid vapors can corrode internal metal components of pipettors and other instruments. Avoid prolonged exposure of equipment to vapors. Use dedicated pipettes for acid handling and rinse removable parts thoroughly after use. Store acids in tightly sealed containers inside a ventilated acid cabinet. Keep away from incompatible materials such as bases and oxidizers.

Step-by-step method details

Preparation of large nuclear extracts

Timing: 4 h

Here, we outline the steps for processing cells from culture and isolating nuclear fractions by swelling and Dounce homogenization. This step is optimized to yield sufficient cell material for downstream immunoprecipitation.

• 1.
  In a sterile tissue culture hood, remove medium from culture dish by gentle aspiration.

  • a.
    Wash cells twice with sterile 1X PBS.
  • b.
    Add 4 mL of trypsin-EDTA to completely cover the 15 cm dish.
  • c.
    Place them at 37°C for 3-5 min.
  • d.
    Check under the microscope if the cells have detached from the plate.

CRITICAL: It is important to ensure that all cells have detached before proceeding to the next step.

• 2.
  Add 4 mL of complete medium to stop the trypsin-EDTA reaction.

  • a.
    Collect all the liquid in a sterile tube. Pool cells from the same condition in 50 mL conical tubes.
  • b.
    Centrifuge the cell suspension for 5 min at 200 x g at RT.
  • c.
    Remove the trypsin-EDTA solution by aspiration.
  • d.
    Mix the cells with fresh medium containing serum. Pool cells again if needed to have one 50 mL conical tube per sample.
• 3.Determine cell concentration using an automated cell counter or hemocytometer.

Note: In this step you can check cell viability using a method such as trypan blue, cell number, and concentration. Cell viability should be > 90% to proceed.

• 4.Centrifuge cells at 800 x g for 10 min at 4°C. Aspirate off media.
• 5.Resuspend cells in 10 mL cold 1X PBS.
• 6.Centrifuge cells at 800 x g for 10 min at 4°C. Aspirate off supernatant.
• 7.Resuspend cells in 10 mL cold 1X PBS and transfer to a 15 mL conical tube.
• 8.Centrifuge cells at 800 x g for 10 min at 4°C. Aspirate off supernatant.
• 9.Add 5 packed cell volumes of pre-chilled Swelling Buffer with protease and phosphatase inhibitors to the samples and resuspend. Mix well.

Note: This can be approximated using a separate 15 mL conical tube. For 6 x 10⁷ HCT116 cells, this was approximately 5 mL Swelling Buffer.

• 10.Add NP-40 to a final concentration of 0.1% and mix again.
• 11.
  Check for cell lysis using Trypan Blue.

  • a.
    Mix 3 μL of homogenate and 3 μL of Trypan Blue on a hemocytometer slide and check under a microscope (damaged cells = blue cells). At this stage, nuclei should still be intact.
• 12.Allow samples to swell for 20 min while rotating at 4°C.
• 13.Centrifuge samples at 800 x g for 10 min at 4°C. Pour off supernatant.
• 14.Add 2 packed cell volumes of pre-chilled Swelling Buffer with protease and phosphatase inhibitors to the samples and resuspend. Mix well.
• 15.Transfer sample to a glass Dounce homogenizer. Homogenize with B pestle for 10-15 strokes.

Note: If using fewer Dounce homogenizers than samples, be sure to clean the homogenizer well between samples.

• 16.Centrifuge samples at 800 x g for 10 min at 4°C.
• 17.Carefully remove supernatant and clear by centrifugation at max speed (e.g., 15,000-20,000 x g) for 1 h at 4°C. This is your cytoplasmic fraction.

Note: This fraction can be dialyzed and stored at −80°C until further needed.

• 18.To the remaining pellet, add 1 of the current packed cell volume of pre-chilled nuclear lysis buffer with protease and phosphatase inhibitors and resuspend well.
• 19.Transfer sample to a glass Dounce homogenizer. Homogenize with B pestle for 10-15 strokes.
• 20.
  Check for nuclear lysis using Trypan Blue.

  • a.
    Mix 3 μL of homogenate and 3 μL of Trypan Blue Solution on hemacytometer slide and check under a microscope (damaged cells = blue cells). At this stage, nuclei should be lysed.
• 21.
  Rotate samples for 30 min at 4°C.

  • a.
    At this step, pre-chill 4 L of Dialysis Buffer to 4°C
• 22.Centrifuge samples at 18,000 x g for 30 min at 4°C.
• 23.Transfer supernatant to a new tube. This is your nuclear fraction.

Pause point: At this point, samples can be flash frozen in liquid nitrogen and stored at −80°C for 2 months.

Dialysis and protein concentration

Timing: 18–24 h

Dialysis is important for exchanging high salt and glycerol to allow for optimal antibody binding to its target protein while maintaining protein solubility and functional complexes for immunoprecipitation. Refer to Slide-A-Lyzer™ Dialysis Cassettes for more detailed information.

• 24.Have prepared 4 L of cold Dialysis Buffer. Add 1 mM BME and 0.2 mM PMSF immediately before use.
• 25.
  Hydrate the dialysis membrane.

  • a.
    Remove the cassette from its pouch and place it in an appropriate size buoy.
  • b.
    Immerse the cassette in Dialysis Buffer for 2-3 min.
  • c.
    Remove the cassette from the Dialysis Buffer and gently blot edge of cassette with a paper towel to remove excess liquid.

Note: Avoid touching the membrane at any point with gloved hands. Do not blot the membrane, only the plastic frame.

Note: Specific cassette molecular weight cutoffs will need to be determined based on the protein of interest. Cassettes come in multiple volume capacities, which should be determined based on the predicted volume from the starting material.

• 26.
  Inject sample into the dialysis membrane.

  • a.
    Fill the syringe with sample, leaving a small amount of air in the syringe.
  • b.
    With the bevel sideways, insert the tip of the needle through one of the syringe ports located at a top corner of the cassette.
  • c.
    Inject the sample slowly. Withdraw air by pulling up on the syringe piston.
  • d.
    Remove the syringe needle from the cassette while retaining air in the syringe.
  • e.
    Repeat these steps until the entire sample is injected into the cassette.

Note: If you must inject more than once, use a different corner of the cassette to add the sample. Leave a small bubble of air in the cassette to allow for easy removal of the sample.

• 27.Attach dialysis membrane to an appropriate size buoy. Place samples in a container holding 4 L of Dialysis Buffer such that the cassettes float at the top of the buffer.
• 28.Place a magnetic stir bar in the Dialysis Buffer. Incubate samples in Dialysis Buffer while mixing at 4°C. Dialysis can occur for 4 h to 18 h.
• 29.
  Remove samples from dialysis membrane.

  Note: Use caution to avoid contacting the needle to the membrane.

  • a.
    Fill the syringe with a volume of air equal to the sample size.
  • b.
    With the bevel sideways, insert the tip of the needle through a syringe port. Inject air slowly into the cassette to separate the membranes.
  • c.
    Turn the syringe so that needle is on the bottom and allow the sample to collect near the port. Withdraw the sample into the syringe.
• 30.
  Quantify protein concentration by BCA. Use the Immunoprecipitation (IP) Buffer as a blank.

  • a.
    Use > 10 mg of nuclear protein, depending on the abundance of the target protein. The final volume of each IP reaction is 4 mL.

IP of target protein

Timing: 6–24 h

Here, we outline the steps for immunoprecipitating the individual protein or protein complexes of interest that contain citrullinated residues. These steps have been optimized to achieve maximal target capture while maintaining protein interactions.

• 31.Dilute sample in IP buffer to 4 mL volume.
• 32.Save 5% of the input if needed for downstream immunoblotting to assess the success of the IP.
• 33.Clear the lysate at 12,000 g for 15 min at 4°C.
• 34.
  Prepare magnetic beads for immunoprecipitation.

  • a.
    Resuspend Protein A or Protein G Dynabeads well by pipetting up and down at least 10 times.
  • b.
    Immediately pipette 120 μL of Protein A or Protein G Dynabeads to pre-chilled 1.5 mL microfuge tubes on ice.
  • c.
    Wash beads with 1 mL of ice-cold IP Buffer containing freshly added protease inhibitors.

    Note: It is not necessary to remove the bead storage liquid before adding IP Buffer.

  • d.
    Rotate beads for 2 min at 4°C. Beads should be fully resuspended.
  • e.
    Place microfuge tubes on a magnetic rack. Allow solution to clear completely on the magnet. Aspirate supernatant.
  • f.
    Repeat washes with 1mL of ice-cold IP Buffer for a total of three washes. Rotate beads for 2 min at 4°C.

    Note: The choice of Dynabeads Protein A or G depends on the antibody species and isotypes. Please refer to the manufacturer’s instructions.

• 35.Add antibody to magnetic beads.

Note: The amount of antibody will depend on the protein target of interest. For p53, use 2 μg of anti-p53 (DO1) per 1 mg of protein.

• 36.Rotate samples at 4°C for 1 h.
• 37.
  Crosslink antibody to beads

  • a.
    Prepare BS³ crosslinker immediately before use, allowing for 250 μl working solution per sample. Prepare 100 mM BS³ in Conjugation Buffer (Stock solution) and dilute with Conjugation Buffer to creat a 5 mM working solution.
  • b.
    Wash the Dynabeads twice in 1 mL Conjugation Buffer. Place on magnet and discard supernatant.
  • c.
    Resuspend the Dynabeads in 250 μL 5 mM BS³.

    Note: Depending on the experiment and antibody, the BS³ working solution may need to be 2.5 mM. Refer to the manufacturer’s references for more recommendations on BS³ optimization.

  • d.
    Incubate at 20-25°C for 30 min with tilting/rotation.
  • e.
    Quench the cross-linking reaction by adding 12.5 μL Quenching Buffer.
  • f.
    Incubate at 20-25°C for 15 min with tilting/rotation.

    Note: Crosslinking antibody to beads may not be required, but is recommended to reduce elution of the antibody in the final sample to minimize contamination of the antibody heavy and light chains in the MS analysis. If the protein of interest is not a similar size to the antibody heavy (50 kDa) or light (25 kDa) chain, this step can be omitted.

• 38.Wash the cross-linked Dynabeads three times with 1 mL IP Buffer. Place on magnet and discard supernatant.
• 39.Add cleared lysate to the beads. At this point, bead/sample solution will be transferred to a 15 mL conical.
• 40.Rotate samples at 4°C for 2-4 h.

Note: This step can be extended to overnight (18 h) incubation.

• 41.Place tubes on magnetic rack. Remove supernatant.

Note: Save the supernatant as flow through to observe IP efficiency. See troubleshooting 2.

• 42.Wash beads with 4 mL of IP Buffer, and incubate for 2-3 min while rotating at 4°C.
• 43.Place tubes on magnetic rack. Remove supernatant.
• 44.Repeat washing steps for a total of 3 washes.
• 45.Resuspend beads pellet in 30-50 μL IgG Elution Buffer (pH 2.8) and incubate at 95°C for 10 min in a thermomixer (rotate at 1200 rpm).

Note: The low pH buffer elution minimizes the release of heavy and light IgG chains that could interfere with the LC-MS/MS analysis of the target protein or protein complexes.

• 46.Place tubes on magnetic rack. Transfer supernatant to a new 1.5 mL microfuge tube.
• 47.Add Tris base to eluate to neutralize the pH (1 μL of 1 M Tris pH 9.5 per 20 μL of elution).

Note: If sample turns yellow after boiling, add more Tris base to restore the blue color.

• 48.Samples are now ready for SDS-PAGE.

CRITICAL: Before running the preparative SDS-PAGE gel for MS analysis, confirm that the IP is successful using Western blot.

SDS-PAGE and colloidal blue staining

Timing: 12–24 h

Here, we describe the steps to isolate the target protein on a Tris-Glycine SDS-PAGE gel and stain the gel with colloidal blue. We have found this to be the most efficient process for isolating target proteins and removing impurities such as detergents and salts that can interfere with downstream protease digestion and LC-MS/MS analysis. If you are interested in protein complexes that may contain multiple citrullinated proteins, we recommend running the SDS-PAGE gel just long enough for all samples to enter the gel (<5 min). If you are targeting a single protein, we recommend running the gel to completion.

• 49.
  For Tris-Glycine precast gels (Novex WedgeWell 4 to 20%, Tris-Glycine, 1.0 mm, Mini Protein Gel, 10-well).

  • a.
    Immediately prior to use: Add 2-mercaptoethanol 1:10 to 6X Laemmli Sample Buffer. Vortex to mix.
  • b.
    Add 6X Laemmli Sample Buffer with 2-mercaptoethanol to each IP reaction to make 1X working concentration.

    Note: The well capacity for this gel system is approximately 60 μL. Adjust accordingly for other gel systems.

  • c.
    Boil samples at 95°C for 10 min.
  • d.
    Microcentrifuge all reactions for 30 s at 18,000 x g at 20-25°C.
  • e.
    Load 6-10 μL of Precision Plus Protein ladder into the first well.
  • f.
    Load samples.

    Note: To prevent keratin contamination, wear gloves and thoroughly rinse all gel units and staining containers with Milli-Q water. If possible, leave an empty lane between samples to avoid potential cross-contamination due to overloading.

  • g.
    Run gels at constant 70-90 V for 2 h at 20-25°C.

    Note: If you are interested in protein complexes, use a fixed-percentage gel (e.g., 10%) and do not run the gel farther than it takes for the sample to fully enter the gel. If you are interested in a single protein target, run the gel to get maximal separation for the target of interest.

• 50.
  Colloidal Blue Staining

  • a.
    Prepare staining buffer in a 15 cm tissue culture dish as provided in the Colloidal Blue Staining Kit. An example for one NuPAGE Novex Tris-Glycine gel is:

    • i.
      55 mL deionized water
    • ii.
      20 mL methanol
    • iii.
      5 mL Stainer B
    • iv.
      20 mL Stainer A

      Note: Prepare just before use. Shake Stainer B well before use. Stainer A and Stainer B can form a precipitate when combined; this should dissolve within 1–2 min of addition.

  • b.
    Shake gel in staining buffer for 3-12 h.
  • c.
    Remove the staining buffer with vacuum aspiration to avoid contact with the gel. Add 200 mL of deionized water per gel. Shake the gel for 7 h – 18 h at 20-25°C.
  • d.
    Gel should have bands fully visible with a clear background (Figure 1). Gels can be stored short term submerged in Milli-Q water at 4°C.

    CRITICAL: Due to the low stoichiometry of citrullination, the target protein must be clearly visible by colloidal blue staining (∼1 μg or more) for comprehensive mapping of the modified sites. Less protein may be acceptable to detect heavily citrullinated sites.

Figure 1.

Colloidal blue stain and excised bands for p53 immunoprecipitated from nuclear extracts with inducible empty vector or PADI4

In-gel protease digestion for LC-MS/MS analysis

Timing: 2 days

This section outlines the bottom-up proteomics procedure for in-gel protease digestion of the samples prior to LC-MS/MS analysis. We recommend performing two separate digests using proteases with different cleavage specificity to improve sequence coverage of the target protein. We used trypsin and chymotrypsin for mapping the p53 citrullination sites in Indeglia et al.¹ See troubleshooting 3 and 4 on how to minimize keratin and polymer contamination.

• 51.Use a clean razor blade to excise the gel bands containing the target protein or the entire stained short gel regions for protein complexes.

Note: Minimize gel volume to improve digestion efficiency. Run duplicate gel lanes for the separate digests. Alternatively, a strongly stained gel band (∼2 μg) can be split into two slices for separate digests using different proteases.

• 52.Use clean forceps to transfer gel slices into separate 0.5 mL microfuge tubes.
• 53.Add 400 μL of destaining solution and incubate at 37°C with gentle shaking for 30 min. Repeat once. Remove solution after each step using vacuum aspiration.

Note: If gel slices remain strongly stained, repeat the destaining step until the color is pale blue.

• 54.Dry the destained gel slices in a vacuum concentrator for ∼30 min.
• 55.Add 100 μL of reducing solution and incubate at 37°C for 15 min with gentle shaking.
• 56.Remove reducing solution using vacuum aspiration.
• 57.Add 100 μL of alkylating solution and incubate at 37°C for 30 min in the dark with gentle shaking.

Note: Iodoacetamide is light sensitive. Cover the incubator with aluminum foil.

• 58.Discard alkylating solution using vacuum aspiration.
• 59.Add 400 μL of Wash Buffer 1 and incubate at 37°C for 15 min with gentle shaking. Discard the supernatant.
• 60.Add 400 μL of Wash Buffer 2 and incubate at 37°C for 15 min with gentle shaking. Discard the supernatant. Repeat Step 59 once more.
• 61.Dry gel slices in a vacuum concentrator for ∼30 min.
• 62.Add 50 μL Protease Digest Solution to rehydrate the gel slices. Incubate at 37°C for 1 h with gentle shaking.

Note: Replace trypsin with chymotrypsin (or another appropriate protease) for the second digest. See troubleshooting 5.

• 63.Add 30 μL Digest Dilution Buffer and incubate at 37°C for 16-18 h with gentle shaking.

Note: If gel volume is large, increase buffer volume to fully cover the gel slices.

• 64.Transfer the supernatant (digests) into autosampler vials.
• 65.Add 50 μL of Extract Solution 1 to the gel slices. Incubate at 37°C for 30 min with gentle shaking. Transfer the extract to the same autosampler vial.
• 66.Add 50 μL of Extract Solution 2 to the gel slices. Incubate at 37°C for 30 min with gentle shaking. Transfer the extract into the same autosampler vial.
• 67.Flash freeze the vials containing both the digest and extracts in dry ice/ethanol bath or liquid nitrogen.
• 68.Dry samples in a vacuum concentrator set at 4°C.

Pause point: Dried samples can be stored at −20°C until ready for LC-MS/MS analysis.

Protocol for LC-MS/MS analysis of proteolytic digests

Timing: 2–3 days

This protocol details the LC-MS/MS workflow for analyzing protease-digested samples, with specific parameters optimized for detecting subtle mass shifts such as citrullination (+0.984 Da) and achieving comprehensive sequence coverage. A high-resolution mass spectrometer with fast scan speed and an ultra-high-pressure liquid chromatography (UHPLC) system are essential for resolving complex peptide mixtures and achieving deep proteomic coverage. The described method utilizes a Thermo Scientific Q Exactive Plus mass spectrometer integrated with a Vanquish Neo UHPLC system.

• 69.Redissolve lyophilized samples in 30 μL of 0.1% TFA in Milli-Q water.
• 70.Place sample vials in the cooled autosampler and set up Xcalibur instrument sequence control to inject 5-10 μL samples onto an Acclaim PepMap 100 C18 trap column (75 μm × 2 cm, 3 mm particle size).

Note: Peptide concentration can be measured with a NanoDrop. The appropriate load for LC-MS/MS analysis using a 75 μm analytical column is ∼1 μg for complex samples, or ∼0.25 μg for purified proteins. The C18 trap column provides an additional online sample cleanup prior to peptide separation.

• 71.
  Separate peptides on the nanoEase M/Z Peptide BEH C18 nanocapillary analytical column (75 μm × 25 cm, 1.7 μm particle size) following the conditions below.

  • a.
    HPLC Solvent A: 0.1% formic acid in Milli-Q water.
  • b.
    HPLC Solvent B: 0.1% formic acid in acetonitrile.
  • c.
    Set flow rate to 200 nL/min and column chamber temperature to 45°C.
  • d.
    Use a step gradient consisting of 4%–30% solvent B over 75 min, 30%–85% solvent B over 5 min, hold at 85% solvent B for 10 min, and re-equilibrate to initial conditions.

Note: For complex samples, extend the gradient for better peptide separation. Run a short 30 min blank gradient (5%–90% solvent B) between samples to minimize carryover.

• 72.
  Analyze the eluted peptides on the mass spectrometer using data-dependent acquisition (DDA; Figure 2).

  • a.
    Acquire full MS scan over an m/z range of 300 to 1900 in positive ion mode at 70,000 resolution, AGC target of 3e6, and maximum ion injection time of 50 ms.
  • b.
    Perform DDA MS/MS scans at 17,500 resolution, AGC target of 1e5, and maximum ion injection time of 100 ms on the 20 most abundant precursor ions exceeding a minimum threshold of 10,000 for every full MS scan.
  • c.
    Set normalized collision energy to 30 and dynamic exclusion to 30 s.
  • d.
    Reject unassigned and >5 charge ions, and set “peptide match” to “preferred”.

Note: The acquisition parameters described are optimized for the Q Exactive Plus mass spectrometer. Adjustments may be necessary when using other instruments. At a full MS resolution of 70,000, the system achieves mass accuracy below 5 ppm, allowing it to distinguish peptides with small mass differences across the entire scan range. For purified or immunoprecipitated proteins, starting the full MS scan at 300 m/z and including +1 charge state ions in MS/MS acquisition will enhance detection of shorter peptides (6 to 7 amino acids), thereby improving protein sequence coverage.

CRITICAL: To maintain mass accuracy, the mass spectrometer must be calibrated at regular intervals (weekly) and before starting a new experimental batch of samples. Regular LC system maintenance is also essential for reproducible gradients and stable retention times, which are required to achieve reliable results across replicates.

Figure 2.

Base peak chromatograms of recombinant p53 digested with trypsin (top) and chymotrypsin (bottom)

Chymotrypsin, due to its lower cleavage specificity compared to trypsin, generates a larger number of peptide fragments. When used in combination with trypsin digestion, chymotrypsin enhances the overall sequence coverage of the p53 protein.

Database search and identification of citrullinated residues

Timing: 1–2 days

This section describes the analysis of MS data using MaxQuant¹² version 2.2.0.0 or later. MaxQuant is a widely used, freely available software program for high-resolution LC-MS/MS data processing. For a comprehensive overview of standard proteomics parameters, refer to Tyanova et al.¹⁴ To detect citrullination post-translational modification (PTM), specific adjustments to the default search parameters are required, particularly when analyzing data from trypsin and chymotrypsin digests. This protocol describes the optimized settings essential for identifying citrullinated peptides and modification sites.

• 73.
  Configure citrullination as a modification.

  • a.
    Navigate to “Configuration” tab and add the parameters for the conversion of arginine to citrulline under “Modifications”.

    • i.
      Name: Citrulline (R)
    • ii.
      Description: Citrullination
    • iii.
      Type: Standard
    • iv.
      Composition: H(-1) N(-1) O (Mass = 0.98402)
    • v.
      Position: Anywhere
    • vi.
      Specificities: R
    • vii.
      Neutral losses name: HCNO
    • viii.
      Neutral losses composition: H C N O (Mass = 43.00581)
• 74.
  Load raw MS data

  • a.
    Navigate to “Raw data” tab.
  • b.
    Load the appropriate raw MS files and complete the experimental details.
  • c.
    Process trypsin raw files separately from the chymotrypsin files.
• 75.
  Set group-specific parameters.

  • a.
    Navigate to “Group-specific parameters” tab and modify the following parameters.
  • b.
    Click on “Modifications”

    • i.
      Variable modifications: Oxidation (M), Acetyl (Protein N-term), Citrulline (R), Deamidation (NQ)
    • ii.
      Fixed modifications: Carbamidomethyl (C)
    • iii.
      Max. number of modifications per peptide: 5
  • c.
    Click on “Label-free quantification”

    • i.
      Label-free quantification: LFQ
  • d.
    Click on “Digestion”

    • i.
      Digestion mode: Specific
    • ii.
      Enzyme: Trypsin/P or Chymotrypsin+

      Note: Select the appropriate protease used if different from the above.

    • iii.
      Max. missed cleavages: 4

      Note: Citrullination renders arginine resistant to trypsin cleavage. Increasing the number of missed cleavages improves detection. For chymotrypsin data, use “Chymotrypsin+” (cleaves after Y, W, F, L, M) for broader coverage than “Chymotrypsin” (cleaves after Y, W, F).

      CRITICAL: Include Deamidation (NQ) as a variable modification to avoid misidentifying citrullination, especially when multiple potential sites are present in a peptide.

• 76.
  Set global parameters.

  • a.
    Navigate to “Global parameters” tab and modify the following parameters.
  • b.
    Click on “Sequences”

    • i.
      Fasta files: Add the appropriate protein database in FASTA format. Must include target protein sequence.
    • ii.
      Include contaminants: Checked
    • iii.
      Min. peptide length: 6
    • iv.
      Max. peptide mass [Da]: 7000

      Note: Adjusting peptide length and mass improves sequence coverage by allowing detection of shorter and longer peptides.

  • c.
    Click on “Identification”

    • i.
      Set PSM, Protein, and Site False Discovery Rates (FDR) to 1% to ensure reliable protein and peptide identifications.
    • ii.
      Min. score for modified peptides: 40
• 77.
  Analyze MaxQuant output.

  • a.
    Open output text files in Microsoft Excel to sort and filter citrullinated proteins, peptides, and sites.
  • b.
    The proteinGroups.txt file contains information on the identified proteins.

    • i.
      The “Citrulline (R) site positions” column indicates the position of citrulline residues on the protein.
    • ii.
      The “LFQ intensity” columns indicate the protein abundance determined by label-free quantification.
  • c.
    The modificationSpecificPeptides.txt file contains information on the identified peptides.

    • i.
      The “Citrulline (R)” column indicates the number of citrulline residues per peptide.
    • ii.
      The “Intensity” columns indicate the peptide abundance.
  • d.
    The Citrulline (R)Sites.txt contains information on the identified citrullination sites.

    • i.
      The “Positions within proteins” column indicates the position of citrulline residues on the protein.
    • ii.
      The “Localization prob” column indicates confidence of citrulline site assignment. Use localization prob > 0.75 for high confidence localization of the citrullination site.
    • iii.
      The “Intensity” columns indicate the citrulline site-specific abundance.

Optional: To further validate the identified citrullinated sites, consider using an alternative MS/MS database search tool such as pFind.¹³

• 78.
  Review the MS/MS spectra of citrullinated peptides to confirm the presence of citrulline-related diagnostic fragment ions (Figure 3).

  • a.
    Navigate to “Visualization” tab and select the peptide sequence to view the annotated MS/MS spectrum. troubleshooting 6.
  • b.
    b and y fragment ions containing a citrullinated residue often exhibit a neutral loss of isocyanic acid, resulting in a characteristic peak at −43.01 Da.
  • c.
    The citrulline immonium ion may be observed at 130.10 Da in MS/MS spectra, serving as an additional diagnostic marker.¹⁵ See troubleshooting 7 and 8.

Figure 3.

Representative MS/MS spectra of citrullinated p53 peptides

(A) MS/MS spectrum of a tryptic p53 peptide containing three citrullinated residues at positions 335, 337, and 342.

(B) MS/MS spectrum of a chymotryptic p53 peptide confirming two of the three citrullination sites identified in the trypsin digest. Citrullinated residues are highlighted in red. The asterisk (∗) indicates fragment ions exhibiting a neutral loss of −43.01 Da.

Expected outcomes

A successful isolation of nuclear fractions from cancer cell lines (10 confluent plates) should yield between 10 mg - 30 mg nuclear protein. This protocol was carried out successfully with 10 mg nuclear protein, although optimization may be necessary depending on the specific experimental conditions.

The described proteomics workflow enables unambiguous identification of citrullination sites on target proteins. The high-resolution mass spectrometer used offers sufficient mass accuracy to distinguish the subtle mass shift of citrullination (+0.9840 Da) from the naturally occurring ¹³C isotope on arginine (+1.0034 Da), which is a common source of false positives with low-resolution instruments.¹⁵ Incorporating multiple proteases with distinct cleavage specificities enhances sequence coverage and provides orthogonal confirmation of citrullination sites (Figure 3). Additionally, including the HNCO neutral loss in the MaxQuant search parameters and detecting a −43.0058 Da shift in b- and y-ion fragments further validates the citrullination events.

Together, these strategies ensure confident and reliable site-specific identification of citrullination modifications in protein samples.

Limitations

The success of this protocol is highly dependent on the availability and performance of an antibody capable of efficiently immunoprecipitating the target protein. Inadequate enrichment of citrullinated proteins will result in poor sequence coverage, leading to missed or low-confidence identification of citrullination sites. Therefore, antibody selection and optimization represent the most critical steps for ensuring effective immunoprecipitation. While this protocol was completed successfully with 6 x 10⁷ cells, the optimal cell number may vary depending on the expected abundance of the target protein, the anticipated frequency of citrullination, and specific sample conditions. Access to next-generation high-sensitivity mass spectrometers, such as the Orbitrap Astral,¹⁶ may reduce the required protein input and allow the analysis of lower abundance targets.

Trypsin is widely used in bottom-up proteomics due to its high cleavage specificity and ability to generate peptides with positively charged C-termini, which enhances ionization efficiency and fragmentation during LC-MS/MS. However, trypsin alone is often insufficient to provide comprehensive protein coverage as the optimal tryptic peptide length for LC-MS/MS analysis typically ranges from 8 to 25 amino acids. To overcome this limitation and achieve a more complete mapping of citrullinated sites, a complementary digestion with an additional protease with distinct cleavage specificity is recommended.

The inability of trypsin to cleave after citrulline serves as an important confirmation of citrullination events. However, differences in tryptic peptide length and ionization efficiency before and after citrullination complicate accurate quantification of the citrullination events. Chymotrypsin can be used for comparative quantitation, but its lower cleavage specificity can result in peptides with ragged ends. Lys-C, which cleaves after lysine and leaves internal arginine or citrulline residues, offers a potential alternative for quantitation. However, Lys-C peptides tend to be longer and may be less detectable by LC-MS/MS, reducing the overall sequence coverage. Depending on the target protein sequence, other proteases may be more appropriate for accurate quantification of citrullination events.

Ultimately, the reliability of citrullination site identification and quantification depends on the efficiency of protein enrichment, the choice of protease used, and the sensitivity of downstream analytical methods.

Troubleshooting

Problem 1

Low protein yields (STEP 30).

Potential solution

If you are experiencing low protein yields, the issue is likely tied to insufficient starting cell numbers (before you begin section STEP 2) or insufficient cell lysis (STEP 9). A practical solution is to increase the amount of starting material to ensure sufficient total protein input. This can be achieved by adding additional 15 cm culture plates to expand the number of cells harvested for lysis. Before collection, verify that the cultures are healthy and at optimal confluency, as low viability or excessive cell death can markedly reduce protein yield and quality. If viability is poor, perform a dead cell removal step (e.g., magnetic bead–based cleanup) to eliminate debris and enrich for viable cells.

Problem 2

Inefficient immunoprecipitation of target protein (STEP 50).

Potential solution

Inefficient immunoprecipitation often stems from suboptimal antibody performance or antibody loss during washes. Start with rigorous antibody optimization: test multiple clones/lots and hosts and verify target recognition by Western Blot. Wash buffers may need to be optimized; save supernatants after IP to determine if the sample is being lost during a wash. Ensure you have chosen beads that match your antibody isotype (Protein A/G). You may consider pre-blocking your beads (5% BSA/Casein) to reduce nonspecific binding. To prevent antibody leaching and heavy/light chain contamination, you may consider crosslinking the antibody to beads using BS3. For this, bind the antibody to Protein A/G beads in PBS (avoid primary amine buffers like Tris during the crosslink step), add BS3 at the recommended concentration and time, then quench with Tris or glycine. Wash beads thoroughly and proceed with IP under more stringent washes.

Problem 3

High keratin contamination (STEP 51).

Potential solution

Keratin contamination is a common and significant problem in MS workflows. Keratins are highly prevalent environmental contaminants (primarily from skin, hair, and clothing) that can easily dominate peptide signals, increasing background noise, and complicating data interpretation. To minimize contamination, always use clean lab coats and gloves. Use a disposable personal protection gown over your lab coat, and cover your hair with a disposable bouffant cap. Thoroughly rinse the gel running apparatus with Milli-Q water, and protect it from airborne dust. Perform in-gel digestion and other sensitive sample handling steps in a dedicated PCR workstation equipped with laminar flow and a HEPA filter to reduce airborne contaminants. Clean all tools (e.g., razor blades, forceps, tweezers) that will contact gels by rinsing thoroughly with methanol, followed by Milli-Q water. Wipe down work surfaces and equipment with methanol followed by water before beginning sample preparation. Include gel blank controls during sample processing to monitor background contamination. Despite the above precautions, complete elimination of contamination is impossible. Therefore, it is necessary to add keratins and other common contaminants in the search database to reduce false positive identifications.

Problem 4

Excessive polymer contamination (STEP 51).

Potential solution

Polymer contamination is most commonly caused by polyethlene glycol (PEG), which is a common contaminant in plasticware, concentrators, solvents, and detergents. During LC-MS/MS analysis, PEG contamination typically appears as intense signals with a repeating mass unit of 44 Da. These strong polymer signals can suppress true peptide signals and interfere with peptide identification. To minimize polymer contamination, rinse microfuge tubes and autosampler vials with 50% methanol/0.1% TFA, followed by a rinse with 100% methanol, and allow to air-dry in the PCR workstation before use. Alternatively, use low-retention, MS-grade certified microfuge tubes and autosampler vials that are free from PEG and other polymer additives. Ensure all solvents used (e.g., acetonitrile, methanol) are LC-MS grade and freshly prepared.

Problem 5

Poor sequence coverage with trypsin or chymotrypsin (STEP 62).

Potential solution

Sequence coverage in bottom-up proteomics is highly dependent on the choice of protease, which determines the peptide fragments available for LC-MS/MS analysis. While trypsin and chymotrypsin are commonly used due to their well-characterized cleavage patterns, they may not always yield optimal coverage for every protein. Perform theoretical protease digestion of your target protein to evaluate expected peptide lengths and coverage. GPMAW¹⁷ or PeptideCutter (web.expasy.org/peptide_cutter/) can be used for this purpose. Ensure proper enzyme-to-substrate ratio, incubation time, and buffer conditions to maximize digestion efficiency.

Problem 6

Inability to export annotated MS/MS spectra from MaxQuant for publication (STEP 78).

Potential solution

MaxQuant provides visualization of MS/MS spectra with fragment ion annotations, but the function to export these annotated spectra for publication does not work. We recommend using pFind¹³ to generate high-quality, publication-ready MS/MS spectra with proper annotation of b- and y-ions, including their respective neutral loss ions.

Problem 7

Difficulty confirming citrullination and site localization by manual inspection of MS/MS spectra (STEP 78).

Potential solution

Manual inspection of MS/MS spectra can occasionally fail to conclusively verify the presence of citrullination, especially in peptides containing multiple arginine (R), asparagine (N), or glutamine (Q) residues. This makes it challenging to confidently localize the citrullinated site. To validate the identity and site of citrullination, synthesize the candidate citrullinated peptide and analyze it using LC-MS/MS under identical experimental conditions. Comparison of the MS/MS spectrum and retention time of the synthetic peptide with the endogenous peptide can confirm both the presence of citrullination and the precise localization of the modified residue.

Problem 8

No citrullinated residues were detected (STEP 78).

Potential solution

If citrullinated residues are not identified by LC-MS/MS, first confirm your target protein is likely citrullinated through an in vitro citrullination assay (see before you begin section). Use immunoblotting with anti-citrulline antibodies to detect citrullination in both in vitro samples and cell lysates. If no signal is observed, the protein may not be a PAD substrate or may be modified at levels below detection thresholds. If citrullination is absent or weak, consider stimulating cells with agents known to activate PADs such as calcium ionophore to increase modification levels. If citrullination is confirmed but still undetectable by MS, increase the amount of starting material if feasible. Alternatively, use high-sensitivity mass spectrometers (e.g., Orbitrap Astral) capable of detecting low-level modifications with high mass accuracy. Finally, chemical enrichment strategies, such as glyoxal-based derivatization,^18,19 can significantly improve detection, especially when post-digest peptide-level enrichment is performed to reduce sample complexity.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hsin-Yao Tang (tangh@wistar.org).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Alexandra Indeglia (aindeglia@bwh.harvard.edu).

Materials availability

We have not generated new materials in this study. All materials are commercially available with catalog numbers provided in the protocol.

Data and code availability

This protocol does not involve the use of any new datasets or codes.

Acknowledgments

We would like to thank Thomas Beer and the Proteomics Facility at the Wistar Institute. We thank Martina Gatto and the Gardini lab for sharing their IP protocols. Graphic design was created using BioRender, for which the authors possess a license. This work was supported by NIH grants R01 CA102184 (Maureen Murphy), F31CA277953 (A.I.), and R50CA221838 (H.-Y.T.). Wistar Shared Resources are supported by NIH P30 CA010815.

Author contributions

A.I. designed, performed, and optimized the immunoprecipitation and in vitro citrullination assays related to this protocol. H.-Y.T. designed, performed, and optimized the analysis and identification of the citrullination residues. A.I. and H.-Y.T. wrote and edited the protocol, supervised the project, and secured funding.

Declaration of interests

The authors declare no competing interests.