µMap Photoproximity Labeling on the Cell Surface

Abstract

Understanding a protein's interactome provides crucial insight into its function and its contribution to disease. Traditional methods such as co‐immunoprecipitations often fail to capture interactions that are dependent on native cellular architecture such as those found on the cellular membrane. While enzyme‐based proximity labeling utilizing peroxidases or biotin ligases can achieve in situ interactome mapping, these approaches are limited by labeling radii, cellular engineering, and amino acid labeling biases. Antibody‐guided µMap photoproximity labeling addresses the constraints of these alternative platforms. Here, we apply µMap photoproximity labeling to study the interactome of HER2 by using antibody‐guided proximity labeling to target the endogenous protein, ablating the need for cellular engineering, and leveraging the advantages of µMap's short 4‐nm labeling radius. The protocols presented here describe the preparation of iridium‐antibody conjugates and its application in studying protein interactomes through mass‐spectrometry based analysis. While HER2 was used as a model in this article, this method is broadly applicable and can be used to study any cell surface protein with an appropriate commercially available antibody. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Preparation and validation of iridium‐antibody conjugates

Basic Protocol 2: Proximity labeling and streptavidin enrichment for mass spectrometry

INTRODUCTION

It has long been established that proteins are localized into defined assemblies, termed microenvironments, which facilitate fundamental processes, such as signal transduction and gene expression (Lundberg & Borner, 2019). Understanding the contents of these microenvironments and how they are altered in diseased states can provide mechanistic insights and new therapeutic avenues (Ruffner et al., 2007). The pre‐eminent method for determining protein–protein interactions is co‐immunoprecipitation, which relies on antibodies to extract a protein of interest and its interactors from cellular lysates (Meyerkord & Fu, 2015). However, protein–protein interactions that are scaffolded by lipids or nucleic acids, such as those found on the cell surface or at chromatin, are often disrupted by the lysis conditions required for solubilization, leaving an incomplete understanding of protein microenvironments in these important cellular domains (Zafra & Piniella, 2022).

Over the last decade, the field of proximity labeling has emerged to provide an alternative approach for the determination of protein interactomes (Milione et al., 2024; Qin et al., 2021). A protein of interest is fused onto an enzyme that catalyzes the generation of highly reactive intermediates upon the application of external stimuli. These reactive intermediates then covalently tag proteins and other biomolecules in the immediate vicinity that can be identified through streamlined “Omics” protocols. The two most used classes of enzymes are based upon peroxidases (HRP, APEX) and biotin ligases (BirA*, TurboID), which produce reactive intermediates with diffusion limits on the orders of 10 to 250 nm, often resulting in long lists of interactors for further investigation (Bosch et al., 2021; Cho et al., 2020; Howarth & Ting, 2008; Lam et al., 2015). Additionally, the reactive species generated by these enzymes display limited amino acid reactivity. Phenoxyl radicals produced by peroxidases react primarily with tyrosine residues, while biotinoyl‐5’‐AMP generated by biotin ligases labels lysine side chains on proteins, resulting in an underrepresentation of proteins lacking these specific amino acids in mass spectrometry analyses (Bosch et al., 2021; Zhang et al., 2024).

Photoproximity labeling has gained increasing popularity as an alternative to enzyme‐based proximity labeling (Knutson et al., 2024). In this approach, antibodies are used to localize photocatalysts, which convert inert probes into reactive intermediates upon visible light irradiation. One such example, called µMap, utilizes an iridium photocatalyst to activate diazirine molecules into short‐lived carbenes (Geri et al., 2020). The high reactivity of carbenes allows for the labeling of neighboring proteins in a residue‐agnostic manner with an extremely short labeling radius of 4 nm, producing a more refined and accurate interactome with fewer false positives and biases (Oakley et al., 2022; Seath et al., 2021). Additionally, antibody‐directed photoproximity labeling provides a faster and more accessible approach relative to enzyme‐based proximity labeling as photocatalysts can be conjugated to commercially available antibodies, circumventing the often lengthy and troublesome process of genetic engineering. While an iridium‐diazirine system is used here, other photocatalyst‐probe pairs, such as flavin‐phenol or chlorin‐diazo, can be substituted for iridium‐diazirine, although the labeling radius will be impacted by such changes and modifications to the protocol will be needed to accommodate the alternative approaches (Bechtel et al., 2022; Lin et al., 2024; Tong et al., 2025).

In this article we describe the preparation of an iridium‐antibody conjugate and the steps to perform a proximity labeling experiment to map the interactome of a target cell surface protein using HER2 as an example. HER2, a receptor tyrosine kinase, is overexpressed in multiple cancers and plays a significant role in disease progression (Cheng, 2024; Majumder et al., 2021). Anti‐HER2 therapeutics have significantly improved patient outcomes, yet a deeper understanding of the HER2 interactome can provide further advancement and development of next‐generation anti‐HER2 therapeutics (Cheng, 2024). Basic Protocol 1 details the preparation and validation of the iridium‐antibody conjugate while Basic Protocol 2 applies the iridium‐antibody conjugates to profile the interactome of the target protein (HER2) through mass spectrometry (MS).

STRATEGIC PLANNING

The protocols presented here provide a method to map the interactome of a target cell‐surface protein. There are several considerations to consider the feasibility of this approach compared to conventional enzyme‐based proximity labeling. The crux of this approach relies on the availability of high quality commercially available antibodies. While many well‐characterized antibodies have been developed, less‐studied proteins may lack suitable antibodies. In such scenarios, conjugation of an epitope tag (i.e., FLAG‐tag) enables the use of well characterized anti‐tag antibodies for iridium conjugation. Target protein expression also strongly influences labeling efficiency and proteomic resolution, with highly expressed proteins typically yielding higher signal‐to‐noise ratios and more robust interactome datasets. Overexpression of the target protein may improve signal‐to‐noise ratios and provide a more reliable dataset. Lastly, while a polyclonal human IgG control was used in this example, a monoclonal IgG control is also recommended. The IgG control should also correspond to the host species of the target‐specific antibody. In this article, flow cytometry was used to verify antibody binding and biotinylation of the iridium‐antibody conjugates. Other methods such as microscopy can also be employed depending on the equipment available to the user and the user's requirements. There are two Basic Protocols presented in this article (Fig. 1). Basic Protocol 1 comprises of the preparation and validation of iridium‐antibody conjugates. It is crucial to complete every section of the protocol to ensure that the optimal parameters are used for mapping the interactome of the target protein in Basic Protocol 2.

Figure 1.

Overview of how antibody guided µMap photoproximity is used to study the interactome of a target protein. Iridium photocatalysts are localized via antibodies to the target protein, converting inert diazirine biotin probes into reactive carbenes upon blue light irradiation at 450 nm. Basic Protocol 1 details the preparation and validation of iridium‐antibody conjugates, which is then assessed for antibody binding and biotinylation using flow cytometry and target enrichment using immunoblotting. Basic Protocol 2 details the procedure for performing proximity labeling and proteomics for mass spectrometry analysis of the target membrane protein interactome.

CAUTION: All reactions must be performed in a suitable fume hood with efficient ventilation. Many of the reactions in this article are highly exothermic; safety glasses and reagent‐impermeable protective gloves should be worn.

NOTE: The iridium photocatalyst and diazirine‐biotin probe are photosensitive compounds. Iridium‐antibody conjugates and diazirine‐biotin probes should be protected from visible light to prevent decomposition of the compounds.

Basic Protocol 1. PREPARATION AND VALIDATION OF IRIDIUM‐ANTIBODY CONJUGATES

This protocol details the preparation and validation of iridium‐antibody conjugates through flow cytometry and immunoblotting. We first detail the preparation of the iridium‐antibody conjugate and the quantification of iridium‐to‐antibody ratio. Optimization of this step is crucial as insufficient photocatalysts will result in a low signal‐to‐noise while excessive conjugation might affect antibody binding. We then describe the validation of antibody binding by flow cytometry and biotinylation with the iridium‐antibody conjugates using flow cytometry and immunoblotting (Fig. 1).

Materials

• Iridium‐DBCO (prepared as described in Oakley et al., 2022; Seath et al., 2023)

• Dimethyl sulfoxide (DMSO)

• Azido‐PEG₂₄‐NHS‐ester (BroadPharm, cat. no. BP‐23452)

• Anti‐human HER2 antibody (Trastuzumab), Clone 4D5‐8 (Leinco, cat. no. LT1500)

• 1 M sodium carbonate (Na₂CO₃) (see recipe)

• Human IgG isotype control (Thermo Fisher Scientific, cat. no. 31154)

• 10% glycerol in Dulbecco's phosphate‐buffered saline (DPBS) (see recipe)

• BCA assay kit (Thermo Fisher Scientific, cat. no. 23225)

• DPBS (Thermo Fisher Scientific, cat. no. 14190‐144)

• A549 cells (ATCC, cat. no. CCL‐185)

• Complete Dulbecco's modified Eagle medium (DMEM) (see recipe)

• TrypLE express (Thermo Fisher Scientific, cat. no. 12604013)

• Trypan blue (Milipore Sigma, cat. no. T8154)

• Bovine serum albumin (BSA) (Sigma‐Aldrich, cat. no. A9647)

• Goat α‐human IgG, Alexa Fluor 647 (Thermo Fisher Scientific, cat. no. A‐21445)

• Diazirine‐biotin (prepared as described in Geri et al., 2020)

• Streptavidin‐Alexa Fluor 647 conjugate (Thermo Fisher Scientific, cat. no. S21374)

• 1× RIPA lysis buffer (see recipe)

• Halt protease inhibitor cocktail (Thermo Fisher Scientific, cat. no. 78429)

• Streptavidin‐coated magnetic beads (Thermo Fisher Scientific, cat. no. 45‐002‐804; Cytiva Life Sciences, cat. no. 28985738)

• 1% SDS in DPBS (see recipe)

• 1 M NaCl (see recipe)

• 10% (v/v) ethanol in DPBS (see recipe)

• Biotin elution buffer (see recipe)

• 4% to 12% Bis‐tris SDS PAGE gel (Thermo Fisher Scientific, cat. no. NW04120BOX)

• 4× Laemmli buffer (Bio‐Rad, cat. no. 1610747)

• 1× MES‐SDS running buffer (see recipe)

• 1× transfer buffer (see recipe)

• H₂O, DI

• Revert 520 total protein stain (Licorbio, cat. no. 926‐10010)

• Revert wash solution (Licorbio, cat. no. 926‐10010)

• Revert destaining solution (Licorbio, cat. no. 926‐10010)

• 1× TBST (see recipe)

• 3% BSA blocking solution (see recipe)

• Anti‐ErbB2 (29D8) rabbit mAb (Cell Signaling Technology, cat. no. 2165)

• IRDYE 800CW goat anti‐rabbit IgG (Licorbio, cat. no. 926‐32211)

• End‐to‐end rotator

• 37°C incubator

• Zeba desalting columns (Thermo Fisher Scientific, cat. no. 89883)

• Microcentrifuge tubes

• Microcentrifuge

• 96‐well clear bottom plate

• Plate spectrophotometer

• Foil

• 15‐cm² tissue culture plates

• Mammalian cell culture incubator, 37°C, 5% CO₂

• Conical tubes

• Hemocytometer or automated cell counter (e.g., Countess)

• Flow cytometry tubes

• Filter caps

• Flow cytometer

• Flow cytometry analysis software (https://www.flowjo.com/flowjo/download)

• M2 photoreactor (Millipore Sigma, cat. no. Z744035)

• Sonicator (Diagenode Bioruptor)

• LoBind tube (Thermo Fisher Scientific, cat. no. 14‐222‐158)

• Magnetic rack (Thermo Fisher Scientific, cat. no. 1232D)

• Heat block

• Nitrocellulose membrane

• Filter paper

• Fluorescent imaging system (e.g., Licor Odyssey Fc Imager)

Synthesis of iridium‐antibody conjugate

This section describes the conjugation of an iridium photocatalyst to an antibody. The process involves the copper‐free click reaction of an iridium photocatalyst bearing a DBCO functional group with an Azido‐PEG₂₄‐NHS ester and the subsequent bioconjugation to an antibody of interest (Fig. 2A). The antibody photocatalyst conjugate is then purified using a desalting spin column and quantified to determine photocatalyst to antibody ratio. This protocol provides experimental conditions that may be used as a reference but should be adjusted depending on the antibody used. The antibody‐photocatalyst conjugate can be stored up to 1 week at 4°C, but we recommend preparing fresh for each experiment, as extended storage may result in precipitation of antibody from solution and decreased efficacy.

Figure 2.

Overview of the synthesis of iridium‐antibody conjugates and visual confirmation of iridium conjugation via UV irradiation. Impact of iridium conjugation on antibody binding is also explored. (A) Synthetic route for conjugation of an iridium photocatalyst to an antibody. An iridium photocatalyst containing a DBCO functional group undergoes a click reaction with azido‐PEG₂₄‐NHS ester to form an iridium‐PEG₂₄‐NHS ester conjugate. This conjugate reacts with lysines on the antibody to form an iridium‐antibody conjugate, with an average of 8 iridium photocatalysts per antibody. (B) Visual confirmation of iridium conjugation via UV irradiation. Iridium conjugated antibodies display visible fluorescence under UV irradiation compared to the unconjugated antibodies. (C) Impact of iridium conjugation on antibody binding. Iridium conjugation shows minimal changes in antibody binding with either IgG or Tz antibodies.

• 1Combine 5 µl iridium‐DBCO (10 mM in DMSO) and 5 µl of freshly resuspended N₃‐PEG₂₄ ‐NHS ester (10 mM in DMSO).

  Iridium‐DBCO photocatalyst can be prepared as described in Oakley et al. (2022).

• 2Rotate at 37°C for 1 hr on a rotator protected from light.
• 3Combine 5 µl of photocatalyst‐PEG₂₄ NHS with 200 µg antibody (100 µl of 2 mg/ml solution recommended) and 2 µl of 1 M Na₂CO₃ (final concentration 20 mM).

  Ensure that the antibody of interest is formulated in carrier‐free solution to avoid non‐specific bioconjugation with carrier proteins. The ratio of antibody to photocatalyst PEG₂₄ ‐NHS‐ester can be adjusted to increase or decrease the final photocatalyst to antibody ratio. This can vary depending on the antibody and may take some optimization. A human IgG is used in this protocol as a negative control.

• 4Incubate for 16 hr on rotator at 4°C protected from light.

  The incubation time between the photocatalyst‐PEG₂₄‐NHS‐ester and antibody can be adjusted to increase or decrease the final photocatalyst to antibody ratio. This can vary depending on the antibody and may take some optimization.

• 5Prepare two Zeba spin columns for each antibody photocatalyst conjugate reaction. Loosen cap of Zeba spin column, remove bottom clog, and place in clean 1.5‐ml microcentrifuge tube. Centrifuge 1 min at 1000 × g, room temperature, and discard flowthrough.
• 6Mark where resin is slanted towards to ensure resin column is oriented in the same direction for subsequent spins.

  Desalting efficiency will be decreased if the tube is not oriented in same direction.

• 7Add 300 µl of 10% glycerol in DPBS to column to equilibrate the column.
• 8Place column back in the 1.5‐ml microcentrifuge tube and centrifuge column 1 min at 1000 × g, room temperature, and discard flowthrough.
• 9Repeat steps 7 and 8 two additional times for a total of 3 washes.
• 10Add antibody PEG₂₄ photocatalyst conjugate to spin column (30 to 130 µl per column).
• 11Place column in clean 1.5‐ml microcentrifuge tube and centrifuge 2 min at 1000 × g, room temperature. Flowthrough is the antibody photocatalyst conjugate.
• 12Transfer antibody photocatalyst conjugate to second Zeba spin column and place in clean 1.5‐ml microcentrifuge tube to ensure complete removal of free photocatalyst.
• 13Centrifuge 2 min at 1000 × g, room temperature. The flowthrough is the purified antibody photocatalyst conjugate.
• 14Quantify protein concentration of antibody photocatalyst conjugate by taking 5 µl of the sample and running a Pierce BCA protein assay. Use protein concentration to calculate molarity of antibody, assuming the standard molecular weight of an antibody is 150 kDa.

  Other methods for protein quantification can also be used.

• 15Add 40 µl of serial dilution of free photocatalyst with known concentrations to a 96‐well clear bottom plate. Generally, a 1:2 serial dilution of photocatalyst from 1 mM to 62.5 µM is adequate to capture the concentration of antibody photocatalyst conjugate within the concentration curve. Add 40 µl of antibody photocatalyst conjugate to one well of the plate (this will be recovered after measuring absorbance).
• 16Measure the absorbance of the plate at 350 nm.
• 17Use the absorbance of the standard curve to calculate concentration of photocatalyst present in antibody photocatalyst conjugate. Recover antibody photocatalyst conjugate. Wrap tube in foil and store at 4°C protected from light until ready for use.

  Antibody photocatalyst conjugate is stable for ∼1 week. It is advised to discard after 1 week.

• 18Determine molar ratio of iridium to antibody by using values determined in BCA and absorbance measurements. Ideal range for iridium to antibody is 5:1 to 10:1.
• 19Visually confirm conjugation of iridium to antibody by placing the conjugate under UV irradiation. Unconjugated antibodies or DPBS can be used as a negative control to assess the baseline fluorescence. Proceed to validate antibody binding by flow cytometry after determining iridium to antibody ratio (Fig. 2B).

Validation of antibody binding by flow cytometry

This section validates that the conjugation of iridium to the antibody has minimal impact to the binding affinity of the antibody to its target protein. Iridium‐conjugated antibodies should display similar binding to the target protein as the unconjugated antibodies, with the IgG antibodies being used as a negative control. Completion of this protocol should provide the optimal concentrations for antibody binding that yields the best signal‐to‐noise compared to the IgG control to be used for the next section validating biotinylation of cells by flow cytometry.

• 20Culture cells in appropriate conditions to be able to collect 4 × 10⁷ cells. In this example, A549 cells are cultured in 15‐cm² tissue culture plates using DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin‐streptomycin and incubated at 37°C and 5% CO₂.
• 21Dissociate cells by washing once with DPBS and then incubating with adequate volume of TryplE Express to cover plate and incubate for 15 min at 37°C.
• 22Collect cells by neutralizing TrypLE with complete growth medium (e.g., DMEM) and transfer to conical tube.
• 23Pellet cells by centrifuging 3 min at 400 × g, 4°C (centrifugation speed may vary depending on cell line). Aspirate medium and resuspend cells in DPBS.
• 24Count cells using trypan exclusion assay. Either manual counting using a hemocytometer or automated counting using a Countess can be used to determine cell concentration.
• 25Aliquot cells into 1.5‐ml microcentrifuge tubes at 2 × 10⁶ cells/tube based on calculations from cell counting. Here we have 17 samples for optimization of antibody concentration.
• 26Pellet cells in 1.5‐ml microcentrifuge tube by centrifugation for 3 min at 400 × g, 4°C.
• 27Resuspend cells in 1 ml of cold DPBS supplemented with 1% BSA. This volume should be adjusted proportionally to the number of cells used.
• 28Add antibody conjugate to each sample. Here we used varying concentrations of conjugated and unconjugated IgG and Trastuzumab (Tz). We used 1, 2.5, 5, and 15 µg/10⁶ cells of each antibody for a total of 16 conditions. We also included an additional tube of no primary antibody to control for non‐specific secondary binding.
• 29Incubate samples at 4°C on a rotator for 30 min.
• 30Pellet cells by centrifuging for 3 min at 400 × g, 4°C.
• 31Carefully aspirate supernatant and resuspend cells in 1 ml of cold DPBS by gentle pipetting to wash cells. Repeat the washes once for a total of two washes.
• 32Resuspend cells in 1 ml of cold DPBS with α‐human‐647 secondary antibody diluted 1:1000.

  Alternative fluorophores on secondary antibody can be used as long as the flow cytometer has the corresponding lasers and filters.

• 33Incubate samples at 4°C on a rotator for 30 min.
• 34Pellet cells for 3 min at 400 × g, 4°C.
• 35To wash cells, carefully aspirate supernatant and resuspend cells in 1 ml of cold DPBS by gentle pipetting. Pellet for 3 min at 400 × g, 4°C. Repeat step once for a total of two washes.
• 36Resuspend cells in 400 µl DPBS and transfer to a flow cytometer tube (ensure compatibility with flow cytometer) by passing through a mesh filter to remove clumped cells.
• 37Run samples on flow cytometer based on manufacturer's instructions. Collect at least 10,000 events with corresponding side scatter, forward scatter, and appropriate channel for fluorophore used.
• 38Analyze flow cytometry data using preferred software (e.g., FlowJo) and quantify number of positive cells for each treatment.
• 39Use data to confirm binding of photocatalyst conjugated antibody to cells at comparable levels of unconjugated antibody and determine concentration that produces best signal‐to‐noise as compared to IgG control. Proceed on to validation of biotinylation of cells by flow cytometry after determining concentration that produces the best signal‐to‐noise (Fig. 2C).

  The ratios of catalyst to antibody and the amount of antibody will vary depending on the cell line, target protein, antibody used, and ratio of photocatalyst to antibody.

Validation of biotinylation of cells by flow cytometry

This section confirms the selective biotinylation of cells with the iridium‐antibody conjugates. The previous section should provide a reference concentration of iridium‐antibody conjugates to start with, although optimization of the various labeling conditions, such as irradiation timing and probe concentration, may be required to obtain an adequate signal‐to‐noise. After completing this protocol, users should know the optimal concentration of iridium‐antibody conjugates to use and the appropriate labeling conditions. These parameters will be used in the next section validating biotinylation of cells by immunoblotting (Fig. 3A).

Figure 3.

Workflow for validating biotinylation using iridium‐antibody conjugates and analysis of proteomics results. (A) Overview of workflow validating biotinylation of target protein using flow cytometry and immunoblotting before performing proteomic analysis. (B) Flow cytometry data displaying an increase in streptavidin signal with iridium‐Tz conjugates relative to iridium‐IgG conjugates. (C) Western blots demonstrating an increase in HER2 signal following streptavidin enrichment with iridium‐Tz conjugates. An increase in streptavidin intensity is also observed, corroborating results observed with flow cytometry. (D) Volcano plot showing interactors of HER2. (E) Gene ontology analysis of HER2 interactors.

• 40Culture cells in appropriate conditions to be able to collect 4 × 10⁷ cells. Here we culture A549 cells in 15‐cm² tissue culture plates using DMEM supplemented with 10% FBS and 1% penicillin‐streptomycin and incubated at 37°C and 5% CO₂.
• 41Dissociate cells by washing once with DPBS and then incubating with adequate volume of TrypLE Express to cover plate and incubate for 15 min at 37°C.
• 42Collect cells by neutralizing TrypLE with complete growth medium (e.g., DMEM) and transfer to conical tube.
• 43Centrifuge cells for 3 min at 400 × g, 4°C (centrifugation speed may vary depending on cell line). Aspirate medium and resuspend cells in DPBS.
• 44Count cells using trypan exclusion assay. Either manual counting using a hemocytometer or automated counting using a Countess can be used to determine cell concentration.
• 45Aliquot cells into 1.5‐ml microcentrifuge tubes at 2 × 10⁶ cells/tube based on calculations from cell counting.
• 46Pellet cells in 1.5‐ml tube by centrifugation for 3 min at 400 × g, 4°C.
• 47Resuspend cells in 1 ml DPBS supplemented with 1% BSA. This volume should be adjusted proportionally to the number of cells used.
• 48Add iridium‐antibody conjugates to each sample at concentration determined to produce the best signal‐to‐noise in antibody binding optimization steps above.
• 49Incubate samples at 4°C on a rotator for 30 min protected from light.
• 50Pellet cells for 3 min at 400 × g, 4°C.
• 51Wash cells by carefully aspirating supernatant and resuspend cells in 1 ml of cold DPBS by gentle pipetting.
• 52Resuspend cells in 1 ml of cold DPBS containing 250 µM diazirine‐biotin.
• 53Irradiate samples for 90 s with 450 nm blue light using a M2 photoreactor.

  Labeling time and probe concentration should be optimized for each experiment to ensure the best signal‐to‐noise as determined by streptavidin signal. Diazirine‐biotin probes can either be purchased (MedChemExpress, cat. no. HY‐154801) or prepared as described in Geri et al. (2020).

• 54Pellet cells for 3 min at 400 × g, 4°C.
• 55Wash cells by carefully aspirating supernatant and resuspend cells in 1 ml of cold DPBS by gentle pipetting. Repeat step once for a total of two washes.
• 56Resuspend cells in 1 ml DPBS with streptavidin‐647 at a 1:1000 dilution. Alternative fluorophores on streptavidin can be used as long as flow cytometer has the corresponding lasers and filters.
• 57Incubate samples at 4°C on a rotator for 30 min protected from light.
• 58To wash cells, carefully aspirate supernatant and resuspend cells in 1 ml cold DPBS by gentle pipetting. Pellet for 3 min at 400 × g, 4°C. Repeat step once for a total of two washes.
• 59Resuspend cells in 400 µl DPBS and transfer to a flow cytometer tube (ensure compatibility with flow cytometer) by passing through a mesh filter to remove clumped cells.
• 60Run samples on flow cytometer based on manufacturer's instructions. Collect at least 10,000 events with corresponding side scatter, forward scatter, and appropriate channel for fluorophore used.
• 61Analyze flow cytometry data using preferred software (e.g., FlowJo) and quantify number of positive cells for each treatment.
• 62Confirm selective biotinylation of cells treated with primary antibody conjugate, ensuring limited background labeling in IgG controls. Proceed onto validation of biotinylation by immunoblotting after parameters have the aforementioned variables have been optimized for adequate signal‐to‐noise (Fig. 3B).

  These values will vary depending on cell line, protein target, antibody, irradiation time, and photocatalyst being used. Optimization of these variables may be required to obtain adequate signal‐to‐noise.

Validation of biotinylation of target protein by immunoblotting

This section validates that the target protein (HER2 in this example) is selectively enriched after streptavidin‐IP. The parameters used in this protocol should follow that used in the previous section validating biotinylation of cells by flow cytometry. In this section we cover the lysis and streptavidin enrichment of biotinylated proteins with confirmation via western blot. Successful proximity labeling by the iridium antibody conjugate should result in an enrichment of the target protein relative to iridium‐IgG in the elution with a corresponding increase in streptavidin intensity.

• 63Culture cells in appropriate conditions to be able to collect 5 × 10⁶ cells. Here we culture A549 cells in 15‐cm² tissue culture plates using DMEM supplemented with 10% FBS and 1% penicillin‐streptomycin and incubated at 37°C and 5% CO₂.

  Different number of cells may have to be used to obtain an appropriate amount of lysate (1 mg of lysate is needed per replicate). Less than 1 mg of lysate for streptavidin enrichment can be used but may result in lower signals or undetectable bands in the western blot.

• 64Repeat steps 41 to 55.
• 65Resuspend the cells in 1 ml of 1× RIPA lysis buffer supplemented with 1× protease inhibitor.
• 66Sonicate the samples on a Diagenode Bioruptor for 12 cycles, 30 s on 30 s off, high, at 4°C.
• 67Centrifuge the samples 15 min at 17,000 × g, 4°C, to clarify lysate.
• 68Transfer the supernatant containing the lysate to a new microcentrifuge tube.
• 69Determine the protein concentration of the lysate via BCA assay or by a method of your choice and normalize protein concentration across all experimental replicates to 1 mg/ml with 1× RIPA lysis buffer.
• 70For the number of samples being used, add 100 µl of magnetic sepharose streptavidin beads to a LoBind tube, place on a magnetic rack, and carefully remove the storage buffer with a pipette.

  If protein yield is <1 mg/ml, adjust the volume of beads to maintain a ratio of 100 µl beads to 1 mg lysate. Using <1 mg of protein may result in lower signal or undetectable bands in the western blot.

• 71Wash the beads gently by resuspending in 1 ml of 1× RIPA lysis buffer, mixing the beads by gently inverting the tube.
• 72Place tube on magnetic rack and remove supernatant. Repeat steps 71 to 72 two additional times.
• 73Resuspend beads in 100 µl of 1× RIPA lysis buffer.
• 74Add 1 ml of each sample to 100 µl prewashed beads and incubate for 2 hr at room temperature or overnight at 4°C with end‐over‐end rotation.
• 75Place tube on magnetic rack and remove supernatant.
• 76Wash the beads gently by resuspending in 1 ml of 1% (w/v) SDS in DPBS, mixing the beads by gently inverting. Repeat steps 75 to 76 two additional times.
• 77Place tube on magnetic rack and remove supernatant.
• 78Wash the beads gently by resuspending in 1 ml of 1 M NaCl in DPBS, mixing the beads by gently inverting. Repeat steps 77 to 78 two additional times.
• 79Place tube on magnetic rack and remove supernatant.
• 80Wash the beads gently by resuspending in 1 ml of 10% ethanol (v/v) in DPBS, mixing the beads by gently inverting. Repeat steps 79 and 80 two additional times.
• 81Place tube on magnetic rack and remove supernatant.
• 82Wash the beads with 1 ml DPBS once and resuspend the beads in 30 µl biotin elution buffer.
• 83Boil at 95°C for 15 min to elute biotinylated proteins.
• 84Briefly spin down the beads using a tabletop centrifuge for 2 s at 2000 × g, room temperature, place tube in magnetic rack, and transfer the supernatant to a new microcentrifuge tube while warm.
• 85Allow the sample to cool down to room temperature before loading onto a 4% to 12% Bis‐Tris SDS page gel.
• 86Combine 20 µg of each sample with 4× Laemmli buffer and heat at 95°C for 10 min to serve as an input control. Let the input control cool to room temperature before loading the input control and the elution on the same gel.
• 87Run the gel in 1× MES‐SDS running buffer at 150 V for 1 hr.
• 88Transfer the protein to a nitrocellulose membrane. Several methods of transfer are available. A wet transfer using transfer buffer was used in this example. Run at 20 V for 2 hr.
• 89Fully dry the membrane by placing it on top of a clean filter paper and incubate at 37°C for 10 min.
• 90Rehydrate the membrane by incubating in DPBS for 5 min at room temperature with gentle shaking.
• 91Rinse the membrane with DI water twice.
• 92Incubate the membrane in 10 ml of 520 total protein stain for 5 min with gentle shaking.
• 93Decant the total protein stain solution and wash the membrane with 10 ml Revert wash solution twice for 30 s with gentle shaking.
• 94Remove the wash solution thoroughly and rinse the membrane with DI water.
• 95Immediately image the membrane in the 520 nm channel. This will serve as a loading control and a verification of the accuracy of protein loading.
• 96Rinse the membrane in DI water.
• 97Incubate the membrane in 10 ml of Revert destaining solution for 5 to 10 min or until bands are no longer visible.

  Do not incubate membrane in Revert destaining solution for >10 min.

• 98Decant the destaining solution thoroughly and wash the membrane with DI water twice.
• 99Rinse the membrane with TBST twice and block for 1 hr at room temperature using 3% BSA in TBST with gentle shaking.
• 100Remove the blocking solution.
• 101Incubate the membrane with the appropriate primary antibody diluted in TBST at 4°C overnight with gentle shaking. A 1:1000 dilution of anti‐ErbB2 (29D8) rabbit mAb was used in this example.
• 102Wash with TBST twice for 5 min with gentle shaking.
• 103Incubate the membrane with the appropriate secondary antibody diluted in TBST for 1 hr at room temperature with gentle shaking. A 1:10000 of IRDYE 800CW goat anti‐rabbit was used in this example.
• 104Wash the membrane with TBST twice for 5 min with gentle shaking and image membrane immediately using a fluorescent imaging system. A clear distinct band corresponding to the protein of interest should appear in the lane corresponding to the iridium‐antibody with minimal background observed in the lane corresponding to iridium‐IgG (Fig. 3C).

Basic Protocol 2. PROXIMITY LABELING AND STREPTAVIDIN ENRICHMENT FOR MASS SPECTROMETRY

Basic Protocol 2 describes the protocol for mass spectrometry analysis of biotinylated proteins. Three biological replicates per condition are used to ensure robust and reproducible results for mass spectrometry analysis, although more replicates can be added if desired by the user. Parameters for this protocol should follow the same conditions used to validate biotinylation via immunoblotting in Basic Protocol 1. Instructions for data analysis are also included in this protocol. The entire protocol should be performed carefully with appropriate personal protective equipment to minimize keratin contamination from the user. Reagents and buffers should be prepared fresh to increase accuracy of results obtained.

Additional Materials (also see Basic Protocol 1)

• 100 mM ammonium bicarbonate (see recipe)

• 3 M urea in DPBS (see recipe)

• Reduction solution (200 mM DTT in 25 mM ammonium bicarbonate) (see recipe)

• Alkylation solution (500 mM IAA in 25 mM ammonium bicarbonate) (see recipe)

• 50 mM ammonium bicarbonate (see recipe)

• MS‐grade trypsin (Thermo Fisher Scientific, cat. no. 90057)

• MS‐grade acetic acid (Thermo Fisher Scientific, cat. no. A1131AMP)

• Proteomics mass spectrometry raw files

• DiaNN (https://github.com/vdemichev/DiaNN/releases)

• Perseus (https://maxquant.net/perseus/)

Proximity labeling and cell lysis

This section describes the proximity labeling of the target protein (HER2 in this example) using iridium‐antibody conjugates (Ir‐Tz). While the workflow for the protocol is similar to that in Basic Protocol 1, it is important to note that three biological replicates are used in this section to obtain adequate numbers for statistical analysis. Additionally, buffers used in this protocol should be prepared fresh on the day of the experiment to ensure highest quality proteomics.

• 1Culture cells in appropriate conditions to be able to collect 15 × 10⁶ cells. Here we culture A549 cells in 15‐cm² tissue culture plates using DMEM supplemented with 10% FBS and 1% penicillin‐streptomycin and incubated at 37°C and 5% CO₂.

  Different number of cells may have to be used to obtain an appropriate amount of lysate (1 mg of lysate is recommended per replicate). Adjust the amount of iridium antibody conjugates as necessary.

• 2Dissociate cells by washing once with room temperature DPBS and then incubating with adequate volume of TrypLE Express to cover plate and incubate for 15 min at 37°C.
• 3Collect cells by neutralizing TrypLE with complete growth medium (e.g., DMEM) and transfer to conical tube.
• 4Centrifuge cells for 3 min at 400 × g, 4°C (centrifugation speed may vary depending on cell line). Aspirate medium and resuspend cells in DPBS.
• 5Count cells using trypan exclusion assay. Either manual counting using a hemocytometer or automated counting using a Countess can be used to determine cell concentration.
• 6Aliquot cells into 1.5‐ml microcentrifuge tubes at 2 × 10⁶ cells/tube based on calculations from cell counting.
• 7Pellet cells in 1.5‐ml tube by centrifugation for 3 min at 400 × g, 4°C.
• 8Resuspend cells in 1 ml of ice cold DPBS supplemented with 1% BSA.

  This volume should be adjusted proportionally to the number of cells used.

• 9Add iridium‐antibody conjugates to each sample at concentration determined to produce the best signal‐to‐noise as validated through flow cytometry and immunoblotting in Basic Protocol 1.
• 10Incubate samples at 4°C on a rotator for 30 min protected from light.
• 11Pellet cells for 3 min at 400 × g, 4°C.
• 12Wash cells by carefully aspirating supernatant and resuspend cells in 1 ml of cold DPBS by gentle pipetting.
• 13Resuspend cells in 1 ml of cold DPBS containing 250 µM of diazirine‐biotin.
• 14Irradiate samples for 90 s with 450 nm blue light using a M2 photoreactor.

  While an irradiation timing of 90 s was used in this example, it should be optimized and follow the conditions used when validating through flow cytometry and immunoblotting.

• 15Pellet cells for 3 min at 400 × g, 4°C.
• 16Wash cells by carefully aspirating supernatant and resuspend cells in 1 ml of cold DPBS by gentle pipetting. Repeat step once for a total of two washes.
• 17Resuspend the cells in 1× RIPA lysis buffer supplemented with 1× protease inhibitor.
• 18Sonicate the samples for 12 cycles, 30 s on 30 s off, high.
• 19Centrifuge the samples 15 min at 17,000 × g, 4°C.
• 20Transfer the supernatant containing the lysate to a new microcentrifuge tube.

Enrich biotinylated proteins and digest proteins on‐bead

Here, biotinylated proteins are enriched by applying labeled cell lysate to streptavidin coated magnetic beads. Unbound proteins are washed away, and biotin labeled proteins are retained. Subsequently enriched proteins are reduced, alkylated, and trypsin digested on beads before mass spectrometry‐based proteomics. A label‐free quantification approached was used in this example. However, other approaches such as Tandem Mass Tagging (TMT) may also be used depending on the user's preferences. All buffers used in this section are prepared fresh.

• 21Determine the protein concentration of the lysate via BCA assay or by a method of your choice and normalize protein concentration across all experimental replicates to 1 mg/ml with 1× RIPA lysis buffer with protease inhibitor.

  It is important that the amount of lysate for each experimental replicate is constant among each sample to ensure robust and reproducible results.

• 22For number of samples used, add 100 µl of magnetic sepharose streptavidin beads per 1 mg protein to a microcentrifuge tube and remove the storage buffer using a magnetic stand.

  Adjust the amount of streptavidin beads used depending on the amount of lysate used, ensuring that 100 µl streptavidin beads are used per 1 mg protein. Using <1 mg lysate may result in a lower number of enriched hits.

• 23Wash the beads gently with 1 ml lysis buffer to mix the beads by gently inverting. Repeat this step twice.
• 24Resuspend beads in 100 µl of 1× RIPA lysis buffer.
• 25Add 1 ml of each sample to 100 µl prewashed beads and incubate overnight at 4°C with end‐over‐end rotation.
• 26Place tube on magnetic rack and remove supernatant.
• 27Wash the beads gently by resuspending in 1 ml of 1% (w/v) SDS in DPBS, mixing the beads by gently inverting. Repeat steps 26 to 27 two additional times.
• 28Place tube on magnetic rack and remove supernatant.
• 29Wash the beads gently by resuspending in 1 ml of 1 M NaCl in DPBS, mixing the beads by gently inverting. Repeat steps 28 to 29 two additional times.
• 30Place tube on magnetic rack and remove supernatant.
• 31Wash the beads gently by resuspending in 1 ml of 10% ethanol (v/v) in DPBS, mixing the beads by gently inverting. Repeat steps 30 to 31 two additional times.
• 32Resuspend the beads in 500 µl DPBS and transfer to a new 1.5‐ml LoBind tube.
• 33Remove the supernatant and wash the beads twice with 500 µl DPBS.
• 34Resuspend the beads in 500 µl of freshly prepared 100 mM ammonium bicarbonate and wash twice with 500 µl of 100 mM ammonium bicarbonate.
• 35Resuspend the beads in 500 µl of freshly prepared 3 M urea in DPBS
• 36Add 25 µl of freshly prepared reduction solution (200 mM DTT in 25 mM ammonium bicarbonate)
• 37Incubate at 55°C for 30 min on an orbital shaker at 700 rpm.
• 38Add 30 µl of freshly prepared alkylation solution (50 mM IAA in 25 mM ammonium bicarbonate)
• 39Incubate for 30 min at room temperature in the dark with end‐over‐end rotation.
• 40Remove the supernatant and wash the beads three times with 500 µl of DPBS, mixing the beads by gently inverting between each wash.
• 41Resuspend the beads in 500 µl of 50 mM ammonium bicarbonate and wash with 500 µl of 50 mM ammonium bicarbonate five times, mixing the beads by gently inverting between each wash.
• 42Transfer the beads to a new protein Lobind tube after the last wash and resuspend the beads in 40 µl of 50 mM ammonium bicarbonate.
• 43Add 1.2 µl of freshly prepared MS‐grade trypsin (1 mg/ml) in 50 mM acetic acid.
• 44Incubate for 16 hr with end‐over‐end rotation at 37°C.
• 45Add 0.8 µl MS‐grade trypsin (1 mg/ml) and incubate the beads for an extra 1 hr at 37°C.
• 46Transfer the supernatant to new LoBind tubes and split each biological replicate equally into two technical replicates.
• 47Submit samples for analysis on a Bruker Tims TOF Pro 2 for DIA mass spectrometry.

Analyze proteomics results

Here we describe the pipeline we use to analyze the proteomic data from a Tims TOF Pro2. If MS/MS is conducted on an alternative instrument, the downstream analysis of the raw data should be conducted with the appropriate software and parameters. For analysis of .d files, DiaNN and Perseus software packages were used. This is not the exclusive way this data can be analyzed, but it is easily accessible due to intuitive graphical interfaces available for these software packages.

• 48Use FASTA file downloaded from UniProt for the appropriate species (e.g., Homo sapiens) and generate in silico spectral library in DIANN 1.8.1 with the settings used for peptide search below. This spectral library will be used for processing raw .d data files.
• 49In DIANN, upload raw .d files, the generated spectral library, and the FASTA file corresponding to the species of cells used. Here we use the Homo sapiens proteome, as this is the source of the A549 cells used.
• 50Select the following parameters under the “Precursor ion generation” section: Trypsin/P for protease, 3 missed cleavages, 3 maximum number of variable modifications, N‐term M excision, C carbamidomethylation, Ox(M), and Ac(N‐term). Set both mass accuracy and MS accuracy to 10.
• 51Select the following parameters under the “Algorithm” section: use isotopologues, MBR, no shared spectra, and Heuristic protein inference. Precursor FDR was set to 1%.
• 52Run DIANN and wait for analysis to be completed.
• 53Open the resulting matrix.pg file in Perseus (v2.0.7.0). Input the intensities as main and the other descriptors as categorical.
• 54Transform the data via Log₂ and annotate rows by treatment.
• 55Fill in missing values using default Perseus settings and perform normalization via median subtraction.
• 56Generate a volcano plot utilizing an unpaired t‐test comparing target against IgG proteome (e.g., Tz vs IgG).

  This will generate a matrix of proteins containing Log₁₀(p‐value), Log₂(FoldChange), and a categorical column indicating a proteins significance based on a calculated false discovery rate. This is the final list used to evaluate the interactome of the target protein. The genes associated with the target will be found on the corresponding side of the volcano plot. For a plot with the x‐axis at Log₂(Tz/IgG) the HER2 interactors will be found on the right side of the plot.

• 57Filter the protein matrix generated by Perseus for hits that meet significance based on the FDR curve and have positive Log₂(FoldChange) for significant interactors of the protein of interest.
• 58Analyze and interpret the gene list using a variety of different approaches. Here we compare the list of significant hits to databases of known interactors (STRING and BioGRID) to validate high confidence hits. Then STRING is used to identify gene ontology terms associated with enriched proteins to further validate the data based on expected results (e.g., membrane proteins) and gain insight to new pathways and relationships that may not have been known.

  Many tools exist to analyze and interpret omics datasets. Alternatives include MetaScape, ClusterProfiler, PantherDB, etc.

• 59A literature review should also be performed to identify novel interactors.
• 60In this example, canonical interactors such as EGFR and MET were identified. Proteins suspected to be associated with HER2 but unvalidated on BioGRID were also identified, such as CD151 and NRP1, highlighting the ability of this protocol to capture both canonical and novel interactors (Fig. 3D and 3E).
• 61Once hits are identified from the dataset, it is imperative that orthogonal approaches are used to confirm the interaction. This can be done through approaches, such as co‐IP, proximity ligation assays, proximity labeling of the hit protein, etc. Furthermore, functional studies must be conducted before biological function can be asserted from the proximity labeling dataset.

REAGENTS AND SOLUTIONS

Acetic acid, 50 mM

Add 29 µl acetic acid (Thermo Fisher Scientific, cat. no. A113) to 10 ml H₂O. Prepare fresh before use.

Alkylation solution (500 mM IAA in 250 mM ammonium bicarbonate)

Dissolve 92 mg iodoacetamide (IAA) (MP Biomedicals, cat. no. 100351) in 750 µl H₂O. Add 250 µl of 100 mM ammonium bicarbonate (see recipe). Prepare fresh before use. Store solution in the dark.

Ammonium bicarbonate, 100 mM

Dissolve 0.395 g ammonium bicarbonate (Sigma‐Aldrich, cat. no. A6141) to 50 ml H₂O. Prepare fresh before use.

Ammonium bicarbonate, 50 mM

Add 20 ml H₂O to 20 ml of 100 mM ammonium bicarbonate (see recipe). Prepare fresh before use.

Biotin elution buffer

• 30 mM biotin (Sigma‐Aldrich, cat. no. B4501)

• 6 M urea (Sigma‐Aldrich, cat. no. U5378)

• 2 M thiourea (Sigma‐Aldrich, cat. no. T8656)

• 2% (w/v) sodium dodecyl sulfate (SDS) (Sigma‐Aldrich, cat. no. 74255) in DPBS

• 1× Laemlli buffer (Bio‐Rad, cat. no. 1610747)

• Prepare fresh before use

BSA blocking solution, 3%

Dissolve 300 mg bovine serum albumin (BSA) (Sigma‐Aldrich, cat. no. A9647) in 10 ml TBST (see recipe). Prepare fresh before use.

Complete DMEM

Add 50 ml fetal bovine serum (FBS; 10% final; Thermo Fisher Scientific, cat. no. SH3007102), 5 ml penicillin‐streptomycin (1% final; Thermo Fisher Scientific, cat. no. 15140122) to 445 ml DMEM (Thermo Fisher Scientific, cat. no. 11965092). Store up to 6 months at 4°C.

Ethanol in DPBS, 10%

Add 50 ml ethanol (Fisher Scientific, cat. no. BP2818) to 450 ml DPBS (Thermo Fisher Scientific, cat. no. 14190‐144). Store up to 6 months at room temperature.

Glycerol, 10%

Add 1 ml glycerol (Sigma‐Aldrich, cat. no. G5516) to 9 ml DPBS (Thermo Fisher Scientific, cat. no. 14190‐144). Store up to 6 months at room temperature.

MES‐SDS running buffer, 1×

Add 50 ml of 20× MES‐SDS buffer (Bioland Scientific, cat. no. MES01‐4litres) to 950 ml H₂O. Store up to 6 months at room temperature.

RIPA lysis buffer, 1×

Add 1 ml of 10× RIPA lysis buffer (Milipore Sigma, cat. no. 20‐188) and 100 µl Halt protease inhibitor cocktail (Thermo Fisher Scientific, cat. no. 78429) to 8.9 ml H₂O. Prepare fresh before use.

Na₂CO₃, 1 M

Dissolve 105.9 mg Na₂CO₃ (Sigma‐Aldrich, cat. no. 54132) in 1 ml H₂O. Store up to 6 months at room temperature.

NaCl, 1 M

Dissolve 58.44 g NaCl (Sigma‐Aldrich, cat. no. 746398) in 1 L DPBS (Thermo Fisher Scientific, cat. no. 14190‐144). Store up to 6 months at room temperature.

Reduction solution (200 mM DTT in 250 mM ammonium bicarbonate)

Dissolve 31 mg dithiothreitol (DTT) (Chem‐Impex, cat. no. 00127) in 750 µl H₂O. Add 250 µl of 100 mM ammonium bicarbonate (see recipe). Prepare fresh before use.

SDS in DPBS, 1%

Dissolve 1 g sodium dodecyl sulfate (SDS) (Sigma‐Aldrich, cat. no. 74255) in 100 ml DPBS (Thermo Fisher Scientific, cat. no. 14190‐144). Store up to 6 months at room temperature.

TBST, 1×

Add 100 ml of 10× TBST (Bioland Scientific, cat. no. TBST01‐10litres) to 900 ml H₂O. Store up to 6 months at room temperature.

Transfer buffer, 1×

Add 100 ml methanol (Sigma‐Aldrich, cat. no. 8222835000) and 100 ml of 10× Towbin (Santa Cruz Biotechnology, cat. no. sc‐24955) to 800 ml H₂O. Store up to 6 months at 4°C.

Urea in DPBS, 3 M

Dissolve 1.8 g urea (Sigma‐Aldrich, cat. no. U5378) in 10 ml DPBS (Thermo Fisher Scientific, cat. no. 14190‐144). Prepare fresh before use.

COMMENTARY

Critical Parameters

Preparation of iridium‐antibody conjugate

There are several crucial parameters to consider for the successful preparation of the iridium antibody conjugate. The azido‐PEG₂₄‐NHS ester should be prepared fresh, preferably with anhydrous DMSO to minimize hydrolysis of the NHS ester. Addition of sodium carbonate is crucial to ensure an efficient rate of reaction between the NHS ester and amines on the antibody. Attention should be given to the quality of the antibodies used. Antibodies should, at minimum, be validated for immunofluorescence and ideally be validated for flow cytometry and western blotting. While an ideal photocatalyst to antibody ratio is between 1:5 and 1:10, a slightly lower photocatalyst to antibody ratio is sometimes acceptable as photocatalyst conjugation is dependent on the number of lysines on the antibody. An excess amount of iridium on the antibody may cause it to precipitate out during the desalting process. The iridium antibody should be remade with either a shorter incubation time or a lower ratio of iridum‐PEG₂₄‐NHS to antibody.

Proximity labeling using iridium‐antibody conjugate

The quality of the diazirine‐biotin probe should be validated before each proximity labeling experiment. It is important that the diazirine‐biotin probes are protected from light and stored at an appropriate temperature to minimize the conversion of diazirine to diazo, increasing background biotinylation. Negative controls without irradiation or containing diazirine‐biotin probes only can be added to account for background biotinylation although it is unnecessary in most cases. Cells should be kept on ice and resuspended in cold DPBS to minimize antibody endocytosis and crosslinking. Irradiation time and probe concentration should be optimized for each experiment, although in most scenarios, an irradiation time of 90 s with 250 µM of diazirine‐biotin probes provides an adequate signal‐to‐noise ratio.

Streptavidin enrichment and mass spectrometry analysis of biotinylated proteins

A RIPA lysis buffer is used in this protocol, which is sufficient for the extraction and lysis of most membrane proteins. A membrane protein extraction kit (Thermo Fisher Scientific, cat. no. 89842) can also be used to enrich the amount of membrane protein in the lysate. Protease inhibitors are important to prevent proteolytic degradation during cell lysis, which might reduce the amount of hits obtained during mass spectrometry analysis. Care should be taken when washing the streptavidin beads to preserve the integrity of the streptavidin beads and minimize material loss. The presence of trypsin could potentially mask several protein peaks, reducing the amount of enriched proteins. In such cases, it would be advantageous to increase the amount of lysate before streptavidin enrichment while lowering the amount of trypsin used.

It is important to note that while the BioGRID interactome provides a list of canonical and validated interactors, it is not an exhaustive and comprehensive list of interactors. All enriched hits should be investigated for any potential interactions or similarities in terms of functions or colocalization with the protein of interest. Other quantitative forms of proteomics can also be considered depending on the needs of the user. While a label‐free approach was shown in this example, other methods such as Tandem Mass Tagging (TMT) (Thermo Fisher Scientific, cat. no. A52045) are also acceptable.

Troubleshooting

See Table 1 for a troubleshooting guide for µMap photoproximity labeling on the cell surface.

Table 1.

Troubleshooting Guide for µMap Photoproximity Labeling on the Cell Surface

Problem	Possible cause	Solution
Low amount of iridium conjugation on antibody	Poor reaction efficiency between iridium‐PEG24‐NHS ester and antibody	Increase the incubation time between iridium‐PEG24‐NHS and antibody
		Increase the ratio of iridium‐PEG24‐NHS to antibody
		Prepare fresh Azido‐PEG24‐NHS ester
Iridium‐antibody conjugates do not bind to cells	Poor protein expression in target cell line	Overexpress target protein through transfection
	Poor antibody quality	Use antibodies that have been validated for flow cytometry
	Excess iridium conjugation affected antibody binding	Decrease iridium‐to‐antibody ratio
Low biotinylation with iridium‐antibody relative to iridium‐IgG	Insufficient biotinylation with iridium‐antibody conjugate	Increase irradiation timing or amount of photocatalyst‐antibody conjugates used
	High non‐specific labeling by iridium‐IgG	Increase number of washes before irradiation with diazirine probes
	Diazirine‐biotin probe is degraded	Perform NMR/LC‐MS analysis of the probe to ensure its integrity and protect probe from light; a probe‐only or no‐light irradiation control can also be used to assess background probe activation
	Poor antibody quality	Ensure photocatalyst is conjugated onto an antibody that has been verified for flow cytometry
	Higher amounts of iridium photocatalyst on iridium‐IgG	Ensure that both iridium‐antibody and iridium‐IgG conjugates have similar iridium‐to‐antibody ratios
	Degradation of iridium‐antibody	Prepare fresh iridium‐antibody conjugates before each experiment
Low protein enrichment after streptavidin enrichment	Biotinylated proteins were not eluted from streptavidin beads	Prepare fresh elution buffer and ensure that beads are incubated at 95°C; remove the supernatant while it is still warm
	Insufficient biotinylation of target protein	Increase irradiation timing or amount of iridium‐antibody conjugates.
	Inefficient streptavidin enrichment	Increase amount of streptavidin beads used
High amount of non‐membrane proteins in mass spectrometry analysis	Poor lysis of membrane proteins	Increase amount of RIPA lysis buffer used and sonication cycles; membrane enrichment can also be performed to facilitate lysis of membrane proteins
	Endocytosis of iridium antibody conjugates	Use ice cold DPBS and keep cells on ice to minimize antibody endocytosis
	Keratin contamination during handling	Use appropriate personal protective equipment to minimize keratin contamination

Understanding Results

Basic Protocol 1 covers the preparation and validation of the iridium‐antibody conjugate. An ideal photocatalyst to antibody ratio would be 1:5 to 1:10; however, ratios out of this range are acceptable depending on the antibody used. Importantly, a similar photocatalyst to antibody ratio should be observed between the antibody and IgG conjugates to obtain robust and reproducible results. Iridium conjugation using an NHS‐ester functional group should have minimal to no impact on the antibody binding as demonstrated by Geri et al. (2020) and Lin et al. (2024) (µMap 2019, multimap 2024).

Biotinylation of cells can be evaluated either by flow cytometry or western blot. Flow cytometry provides a quicker readout compared to immunoblotting where iridium‐antibody conjugates should provide a noticeable increase in streptavidin signal relative to the iridium‐IgG conjugates. A modest increase in biotinylation observed in flow cytometry typically translates into a higher signal‐to‐noise in western blotting as the biotinylation observed with iridium‐IgG conjugates are largely non‐specific while the biotinylation observed with iridium‐antibody conjugates are localized to the protein of interest. A clear enrichment of the target protein in immunoblotting is crucial in obtaining good mass spectrometry results.

The target protein should be one of the most enriched hits in mass spectrometry. Subcellular localization of significantly enriched hits (proteins above the FDR curve of 0.05) should be performed to verify the robustness of the experiment, with majority of the hits being membrane associated proteins. A database of protein subcellular localization can be obtained from the human protein atlas (https://www.proteinatlas.org/humanproteome/subcellular). Gene ontology analysis can also be performed as an alternative (https://geneontology.org/). The enriched hits should comprise of both canonical and novel interactors of the target protein. Canonical interactors can be compared against the BioGRID interactome while a literature review of the other hits will have to be performed to validate previous hypotheses or uncover previously unknown interactors.

Time Considerations

The preparation of the iridium‐antibody conjugate can be completed in 18 hr. The validation of antibody binding and biotinylation of cells by flow cytometry can each be completed within a day once cells are confluent; however, a few rounds of optimization might be needed. Validation of biotinylation of target protein by immunoblotting can be completed in 2 to 3 days. The entirety of Basic Protocol 1 can be completed within 1 to 2 weeks depending on how much optimization needs to be performed. In Basic Protocol 2, biotinylated peptides can be prepared and submitted for mass spectrometry analysis in 3 days. Analysis of the proteomic results can take up to 2 days depending on the number of samples and speed of the software.

Author Contributions

Hong Kai Ng: Formal analysis; investigation; methodology; validation; writing—original draft; writing—review and editing. Cameron Douglas: Data curation; formal analysis; investigation; methodology; writing—original draft; writing—review and editing. Ciaran Seath: Conceptualization; project administration; supervision; writing—original draft; writing—review and editing.

Conflict of Interest

The authors declare no conflict of interest.

Supporting information

Excel file containing HER2 proximity proteomics data.

Ng, H. K. , Douglas, C. J. , & Seath, C. P. (2025). µMap photoproximity labeling on the cell surface. Current Protocols, 5, e70216. doi: 10.1002/cpz1.70216

Data Availability Statement

See Supporting Information for data that support this protocol.