Abbreviations used in the protocol are listed in Supplementary Table 1.
1. Preparation of solutions and buffers
NOTE: The commercial details of all the reagents are listed in the Table of Materials.
<ol>
	<li>Prepare 0.1 M Tris-HCl, pH 8.0 solution by adding 1 mL of 1 M Tris-HCl, pH 8.0 into 9 mL of deionized water in a glass vial, and mix well by vortexing. Store at 4 &#176;C for up to 3 months.</li>
	<li>Prepare 24 mM sodium deoxycholate (SDC) by dissolving 10 mg of SDC in 1 mL of 0.1 M Tris-HCl, pH 8.0.</li>
	<li>Prepare 24 mM sodium lauroyl sarcosinate (SLS) by dissolving 7 mg of SLS in 1 mL of 0.1 M Tris-HCl, pH 8.0.</li>
	<li>Prepare elution buffer by mixing 24 mM SDC and 24 mM SLS at a 1:1 (v/v) ratio.</li>
	<li>Prepare 50 ng/&#181;L of trypsin solution by adding 400 &#181;L of deionized water into 20 &#181;g trypsin.</li>
	<li>Prepare 25 mg/mL dithiothreitol (DTT) by adding 308 &#181;L of 0.1 M Tris-HCl, pH 8.0, into 7.7 mg of DTT.</li>
	<li>Prepare 0.25 M iodoacetamide (IAM) by adding 1.2 mL of 0.1 M Tris-HCl, pH 8.0, into 56 mg of IAM.</li>
	<li>Prepare 10% TFA by adding 1 mL of trifluoroacetic acid (TFA) into 9 mL of deionized water. Vortex for 4 s and spin down. Store at 4 &#176;C for up to 3 months.</li>
	<li>Prepare Buffer A by adding 1 mL of 10% TFA into 99 mL of deionized water.</li>
	<li>Prepare Buffer B by adding 1 mL of 10% TFA, and 49 mL of deionized water into 50 mL of acetonitrile (ACN).</li>
	<li>Prepare 10 mM Histidine buffer by adding 37 mg of L-histidine and 54.8 mg of L-Histidine monohydrochloride monohydrate into 50 mL of water.</li>
</ol>
2. Preparation of monoclonal antibody (mAb) solutions
<ol>
	<li>For drug products with concentrations above 50 mg/mL, dilute 15 mg of the monoclonal antibody (mAb) (see Table of Materials) with deionized water in a 2 mL microcentrifuge tube, resulting in a total volume of 300 &#181;L and a final pH of ~6. If necessary, adjust the pH using acetic acid or Tris-HCl.</li>
	<li>For drug products with concentrations below 50 mg/mL, perform buffer exchange following steps 2.2.1 to 2.2.5.
	<ol>
		<li>Transfer 20 mg of each sample to a 10k centrifugal filter device (see Table of Materials) and centrifuge at 14,000 x g for 25 min (at room temperature, RT) until approximately 100 &#181;L volume remains.</li>
		<li>Add 350 &#181;L of 10 mM Histidine (see Table of Materials), pH 6.0 buffer into the filter, vortex, and centrifuge at 14,000 x g for 25 min at RT. Repeat once.</li>
		<li>Invert the filter and place it into the sample collection tube, then centrifuge the samples at 1000 x g for 5 min at RT to retrieve the entire volume in the collection tube. Wash the filter with 200&#160;&#181;Lof 10 mM&#160;Histidine buffer and pipet up and down to recover any remaining protein samples. Transfer the entire solution into the same collection tube, vortex, and spin down.</li>
		<li>Measure the sample concentration by NanoDrop. Adjust the concentration of each protein sample to 50 mg/mL by adding the Histidine buffer prior to incubation with enrichment beads.</li>
		<li>Transfer 300 &#181;L of the sample into a 2 mL microcentrifuge tube.</li>
	</ol>
	</li>
</ol>
3. Preparation of proteome enrichment beads
<ol>
	<li>Remove the top and bottom cap from each spin column obtained from the commercial protein enrichment kit (see Table of Materials). Do not discard the caps, as they will be used later.</li>
	<li>Take the spin column and position it in a 2 mL microcentrifuge tube without a cap. Centrifuge the setup at room temperature and 1,000 x g for 30-60 s at RT in order to eliminate the storage solution. Dispose of the material collected during this step.</li>
	<li>Add 200 &#181;L wash buffer to the commercially available enrichment beads (see Table of Materials) and pipet up and down several times. Place the spin column in a 2 mL microcentrifuge tube and centrifuge at 1,000 x g for 30-60 s at RT to remove the buffer. Discard collected material. 
	NOTE: The wash buffer is included in the commercial protein enrichment kit.</li>
	<li>Repeat steps 3.3 twice.</li>
	<li>Replace the bottom cap and add 200 &#181;L water, then replace the top cap.</li>
</ol>
4. Protein enrichment
<ol>
	<li>Pipet up and down the slurry (from step 3.5) and transfer 40 &#181;L of slurry to the sample prepared in step 2. Incubate and rotate the tube at room temperature for 2 h.</li>
	<li>Make a tip with frit (see Table of Materials) using the 16 G needle to get the appropriate frit size, then insert it into the tip end of the 200 &#181;L tip.</li>
	<li>Transfer the sample with beads to the tip prepared in step 4.2. Centrifuge the tip with 2 mL microcentrifuge tube at 200 x g for 3 min at RT to remove the solution.</li>
	<li>Wash the beads by adding 200 &#181;L wash buffer to the tip and centrifuge the tip with a 2 mL microcentrifuge tube at 200 x g for 2 min at RT to remove the washing solution. Repeat the step twice.</li>
	<li>Wash the beads by adding 200 &#181;L water to the tip and centrifuge the tip with a 2 mL microcentrifuge tube at 200 x g for 2 min at RT to remove the water.</li>
	<li>Elute proteins from beads by adding 10 &#181;L of elution buffer (step 1.4) into the tip, and pipet the slurry ten times in the tip to ensure the beads are soaked with elution buffer.</li>
	<li>Centrifuge the tip with a new 0.5 mL microcentrifuge tube at 200 x g for 30 s at RT to collect the eluent. Repeat steps 4.6-4.7 twice and combine all elution into one 0.5 mL microcentrifuge tube.</li>
	<li>Add 1.5 &#181;L of trypsin solution (step 1.5) into eluent for overnight digestion at 28 &#176;C. Add 1.5 &#181;L of 25 mg/mL DTT (step 1.6), and heat the sample at 90 &#176;C for 20 min.</li>
	<li>Add 1.5 &#181;L of 0.25 M IAM (step 1.7) and incubate at room temperature in the dark for 20 min. Add 3.5 &#181;L 10% TFA (step 1.8), vortex for 2 min, and ensure pH is at ~2-3.</li>
	<li>Centrifuge at 14,400 &#215; g for 10 min at RT to precipitate SDC and SLS. Collect the supernatant for desalting.</li>
	<li>Perform desalting following the steps below.
	<ol>
		<li>Add 50 &#181;L of Buffer B (step 1.10) to the GC-desalting tip (see Table of Materials). Centrifuge the tip with a holder at 1000 x g for 3 min at RT. Discard the collected material.</li>
		<li>Add 50 &#181;L of Buffer A (step 1.9) to the tip. Centrifuge the tip with a holder at 1000 x g for 3 min at RT. Discard the collected material.</li>
		<li>Add the acidifed sample to the tip. Centrifuge the tip with a holder at 500 x g for 6 min at RT. Discard the collected material.</li>
		<li>Wash the tip by adding 50 &#181;L of Buffer A to the tip. Centrifuge the tip with the holder at 500 x g for 3 min at RT. Discard the collected material. Repeat once.</li>
		<li>Elute the peptide from the tip by adding 50 &#181;L of Buffer B to the GC-desalting tip. Centrifuge the tip with a new tube at 500 x g for 3 min at RT. Collect the material. Repeat the step once and combine the eluent.</li>
		<li>Dry the eluent with a vacuum concentrator (see Table of Materials).</li>
	</ol>
	</li>
	<li>Resuspend the dried eluent into 30 &#181;L of 0.1% formic acid (FA) solution.</li>
	<li>Measure the UV of peptide mixtures at 214 nm by a spectrophotometer (see Table of Materials). The concentration of peptide mixtures should range between 0.1 and 0.5 mg/mL.</li>
	<li>Transfer 10 &#181;L of each digested sample into an LC sample vial, then inject 1 &#181;g of each digested sample onto nano LC-MS/MS (see Table of Materials). Perform the analysis following step 5. Store the rest of digested samples in a -80 &#176;C freezer.</li>
</ol>
5. Nano LC-MS/MS analysis
<ol>
	<li>Inject the peptide mixture into a nano-LC system coupled to a mass spectrometer. Load the peptide mixtures (~1 &#181;g) onto a C18 trap column with the gradient provided in Table 1A for desalting and then onto a C18 analytical column at 40 &#176;C for separation. 
	NOTE: The mobile phase A buffer consisted of 0.1% FA in ultra-pure water, and the mobile phase B consisted of 0.1% FA in 80% ACN.</li>
	<li>Separate the peptides and elute with the gradient provided in Table 1B with a flow rate of 250 nL/min. 
	&#8203;NOTE: The mass spectrometer was operated in data-dependent mode (DDA) with a cycle time of 3 s. Ions underwent fragmentation through higher-energy collisional dissociation (HCD) with a normalized collision energy of 30% for each full MS scan. The scans were performed at a resolution of 60,000, with an automatic gain control (AGC) target of 3e6, a maximum injection time of 20 ms, and an m/z range of 380-1600. MS/MS events were conducted at a resolution of 15,000, with an AGC target of 1e5, a maximum injection time of 60 ms, and an m/z range of 200-2000. The exclusion duration was set to 45 s. The ion source properties are provided in Table 1C, and the specific mass spectrometry parameters can be found in Table 2A-F.</li>
</ol>
6. Data analysis
<ol>
	<li>Perform a search of the NISTmAb mass spectrometry raw files against the UniProt mus+musculus database29. Additionally, search for the recombinant protein spiked-in mAb DS mass spectrometry raw files against the UniProt Cricetulus Griseus database30. 
	NOTE: These searches were conducted in Proteome Discoverer software (see Table of Materials) using the Sequest HT and Mascot search engines. The databases used were free of redundant entries.</li>
	<li>For the mass spectrometry searches, apply a mass tolerance of 10 ppm, and set the fragment mass tolerance to 0.02 Da. 
	NOTE: The search criteria encompassed static carbamidomethylation of cysteine residues (+57.0214 Da) and variable modification of oxidation (+15.9949 Da) on methionine residues. Additionally, a variable modification of deamination (+0.984 Da) was applied.</li>
	<li>Perform the database search using trypsin digestion, allowing for a maximum of two missed cleavages. Set the false-discovery rates for both proteins and peptides at 0.01. 
	NOTE: To positively identify host cell proteins (HCPs), a minimum requirement of at least two unique peptides was applied. Table 3 provides the representative summary of identified HCPs associated with NIST mAb analyzed by Proteome Discoverer software.</li>
</ol>

Enrichment of Host Cell Proteins in Monoclonal Antibody Drug Products

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The authors have no competing financial interests.

There are two versions of commercially available protein enrichment beads: one with a smaller capacity and the other with a larger capacity (see Table of Materials). Both versions of the enrichment beads contain ten preps in the package. The manufacturer's instructions suggest that each prep from the small capacity kit can be used to enrich 10 mg of total protein. However, for optimal performance of host cell protein (HCP) enrichment from DS, each prep is good for five DS samples. Therefore, each kit can...

Host cell proteins (HCPs) are impurities that are released from the cell culture of the host organism and co-purified with monoclonal antibody (mAb)1,2,3,4. Trace levels of HCPs can negatively impact the quality of the drug product5,6,7,8,9,10,11,12,13,14,15, and therefore, a sensitive HCP analysis method is desired to detect HCPs in sub-ppm to ppm levels.
Orthogonal methods can be applied to detect HCPs in low abundance. Enzyme-linked immunosorbent assay (ELISA) is generally used to quantitate overall HCPs, and it can also detect and quantitate individual HCPs if the corresponding antibodies are available16. However, the production of HCP-specific antibodies is time-consuming and labor-intensive. In contrast, liquid chromatography coupled with mass spectrometry (LC-MS) can provide comprehensive information about individual HCPs in mAb drug products and is widely applied for HCP identification4,7,9,10,12,13,14,15,17,18,19,20,21,22,23,24,25,26,27.
Several methods have been developed to detect HCPs with LC-MS/MS, including limited digestion20, filtration17, Protein A deletion21, immunoprecipitation (IP), and ProteoMiner enrichment (PM)18. Most methods aim to reduce the amount of mAb and enrich HCPs prior to LC-MS/MS analysis, thereby decreasing the dynamic range between mAb peptides and HCP peptides. This protocol presents a proteomic sample enrichment method that combines ProteoMiner technology and limited digestion (PMLD)28. The ProteoMiner enrichment principle involves using commercially available proteome enrichment beads containing a diverse library of combinatorial peptide ligands. These ligands specifically bind to proteins on antibody-drug products, allowing for the removal of excess molecules while concentrating low-abundance host cell proteins (HCPs) on their respective affinity ligands. On the other hand, the principle of limited digestion involves using a low concentration of trypsin. This concentration is sufficient to digest low-abundance HCPs but not enough to digest all antibody drug products. This approach enables the recovery and enrichment of digested HCP peptides from the solution.
Compared to filtration methods, the PMLD technique is not limited by the size of the detected HCPs17. Protein A deletion methods are specific to detecting HCPs associated with antibodies21, while immunoprecipitation is restricted to predefined HCPs from a particular cell line (such as the Chinese Hamster Ovary (CHO) cell line), where an anti-HCP antibody was generated4. In contrast, PMLD can be applied to detect HCPs from any drug modules and host cell proteins co-purified with drug products from various cell lines. Additionally, PMLD exhibits better sensitivity compared to the mentioned methods17,18,20,21,24.
This approach can enrich the HCP concentration by 7000-fold and lower the detection limit to 0.002 ppm28. The experimental setup is illustrated in Figure 1.

<table><tbody><tr><td>16 G, Metal Hub Needle, 2 in, point style 3</td><td>Hamilton</td><td>91016</td><td></td></tr><tr><td>Acclaim PepMap 100 C18 trap column (20 cm &amp;times; 0.075 mm)</td><td>Thermo Fisher</td><td>164535</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher-Scientific</td><td>A955</td><td></td></tr><tr><td>Acetonitrile with 0.1% Formic Acid (v/v), Optima LC/MS Grade&amp;nbsp;</td><td>Fisher-Scientific</td><td>LS120-4</td><td></td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit</td><td>Millipore Sigma</td><td>UFC5010</td><td></td></tr><tr><td>C18 analytical column (0.075 mm &amp;times; 1.7 &amp;mu;m &amp;times; 30 cm, 100 &amp;Aring;)</td><td>CoAnn Technologies</td><td>HEB07503001718I</td><td></td></tr><tr><td>Centrifuge 5424</td><td>Eppendorf</td><td>5405000646</td><td></td></tr><tr><td>Dithiothreitol (DTT)&amp;nbsp;</td><td>Thermo Fisher</td><td>A39255</td><td></td></tr><tr><td>Frit for SPE cartridges, 9.5 mm, 3 mL, 100/pk</td><td>Agilent</td><td>12131020</td><td></td></tr><tr><td>GL-Tip GC</td><td>GL Sciences Inc&amp;nbsp;&amp;nbsp;</td><td>7820-11201</td><td></td></tr><tr><td>in-house mAb</td><td>Regeneron</td><td></td><td>concentration 200 mg/mL</td></tr><tr><td>Iodoacetamide (30 x 9.3 mg)</td><td>Thermo Fisher</td><td>A39271</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher-Scientific</td><td>149320025</td><td></td></tr><tr><td>L-Histidine</td><td>Sigma Aldrich</td><td>H6034</td><td></td></tr><tr><td>L-Histidine monohydrochloride monohydrate</td><td>Sigma Aldrich</td><td>53370</td><td></td></tr><tr><td>Methanol</td><td>Fisher-Scientific</td><td>A456-4&amp;nbsp;</td><td></td></tr><tr><td>Milli-Q</td><td>Millpore</td><td>30035</td><td></td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td>ND-2000</td><td></td></tr><tr><td>Orbitrap Exploris 480</td><td>Thermo Fisher</td><td>BRE725539</td><td></td></tr><tr><td>Protein LoBind Tube 0.5 mL</td><td>Eppendorf (VWR)</td><td>22431064</td><td></td></tr><tr><td>Protein LoBind Tube 2.0 mL</td><td>Eppendorf (VWR)</td><td>22431102</td><td></td></tr><tr><td>Proteome Discoverer software 2.4</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>ProteoMiner Protein Enrichment Large-Capacity Kit</td><td>Bio-Rad</td><td>1633007</td><td></td></tr><tr><td>ProteoMiner Protein Enrichment Small-Capacity Kit</td><td>Bio-Rad</td><td>1633006</td><td></td></tr><tr><td>Sodium deoxycholate (SDC)</td><td>Sigma Aldrich</td><td>D6750</td><td></td></tr><tr><td>Sodium lauroyl sarcosinate (SLS)&amp;nbsp;</td><td>Sigma Aldrich</td><td>L5777</td><td></td></tr><tr><td>SpeedVac</td><td>Labconco</td><td>7970010</td><td></td></tr><tr><td>Thermomixer R</td><td>Eppendorf</td><td>22670107</td><td></td></tr><tr><td>Trifluoracetic acid (TFA)</td><td>Fisher-Scientific</td><td>28904</td><td></td></tr><tr><td>Trypsin (Sequencing Grade Modified)&amp;nbsp; (5 x 20 ug)</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>Tube Revolver Rotator</td><td>Thermo Fisher</td><td>88881001</td><td></td></tr><tr><td>UltiMate 3000 RSLC nano system</td><td>Thermo Fisher</td><td>ULTIM3000RSLCNANO</td><td></td></tr><tr><td>UltraPure 1 M Tris-HCl pH 8.0</td><td>Thermo Fisher</td><td>15568-025</td><td></td></tr><tr><td>Vortex Genie 2</td><td>VWR</td><td>102091-234</td><td></td></tr><tr><td>Water with 0.1% Formic Acid (v/v), Optima LC/MS Grade&amp;nbsp;</td><td>Fisher-Scientific</td><td>LS118-4&amp;nbsp;</td><td></td></tr></tbody></table>

host cell protein analysis using enrichment beads coupled with limited digestion

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks associated with drug products, methods have been developed to identify low-abundance HCPs. A crucial approach for developing a sensitive HCP detection method involves enriching HCPs while simultaneously removing monoclonal antibodies (mAbs) before analysis, utilizing liquid chromatography-mass spectrometry (LC-MS).
This protocol offers detailed instructions for enriching host cell proteins using commercially available proteome enrichment beads. These beads contain a diverse library of hexapeptide ligands with specific affinities for different proteins. The protocol also incorporates limited digestion and subsequent peptide detection using nano LC-MS/MS. By employing these techniques, HCPs with low abundance can be enriched over 7000-fold, resulting in an impressive detection limit as low as 0.002 ppm. Significantly, this protocol enables the detection of 850 HCPs with a high level of confidence using a NIST mAb. Moreover, it is designed to be user-friendly and includes a video demonstration to assist with its implementation. By following these steps, researchers can effectively enrich and detect HCPs, enhancing the sensitivity and accuracy of risk assessment for drug products.

This protocol presented a sample preparation workflow, termed protein enrichment coupled with limited digestion (PMLD), for the analysis of host cell proteins (HCPs) in a monoclonal antibody (mAb) sample. Figure 1 illustrates the step-by-step procedure of PMLD. The researchers compared the results of HCP analysis using direct digestion (shown in the top panel of Figure 2) and PMLD (shown in the bottom panel of Figure 2). The Total I...

Watch this Scientific Journal Video about Detecting Low-Abundant Host Cell Proteins in Drug Products Using Enrichment Beads and Limited Digestion at JoVE.com

Author Spotlight: Detecting Low-Abundant Host Cell Proteins in Drug Products Using Enrichment Beads and Limited Digestion 

A protocol is presented for enriching host cell proteins (HCPs) from drug products (DP) and detecting peptides using proteome enrichment beads. The method is demonstrated using an in-house manufactured monoclonal antibody (mAb) drug substance (DS), which is a well-characterized reference material for evaluating and comparing different methods in terms of performance.

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks ...

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Pharmacokinetics and Pharmacodynamics

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Unit 1

Pharmacokinetics: Drug Distribution and Protein Binding

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2.1K Views.  Regeneron Pharmaceuticals Inc.. A protocol is presented for enriching host cell proteins (HCPs) from drug products (DP) and detecting peptides using proteome enrichment beads. The method is demonstrated using an in-house manufactured monoclonal antibody (mAb) drug substance (DS), which is a well-characterized reference material for evaluating and comparing different methods in terms of performance.

Video: Author Spotlight: Detecting Low-Abundant Host Cell Proteins in Drug Products Using Enrichment Beads and Limited Digestion 

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Biochemistry

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Biology

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol ...

Neuroscience

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

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