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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#215; 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&#160;</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 &#215; 1.7 &#956;m &#215; 30 cm, 100 &#197;)</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)&#160;</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&#160;&#160;</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&#160;</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)&#160;</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)&#160; (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&#160;</td>
			<td>Fisher-Scientific</td>
			<td>LS118-4&#160;</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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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 

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that belongs to the Direct-Injection Mass Spectrometric (DIMS) technologies. These techniques allow the rapid determination of volatile organic compounds (VOCs), assuring high sensitivity and accuracy. In general, PTR-MS requires neither sample preparation nor sample destruction, allowing real time and non-invasive analysis of samples. PTR-MS are exploited in many fields, from environmental and atmospheric chemistry to medical and biological sciences. More recently, we developed a methodology based on coupling PTR-ToF-MS with an automated sampler and tailored data analysis tools, to increase the degree of automation and, consequently, to enhance the potential of the technique. This approach allowed us to monitor bioprocesses (e.g. enzymatic oxidation, alcoholic fermentation), to screen large sample sets (e.g. different origins, entire germoplasms) and to analyze several experimental modes (e.g. different concentrations of a given ingredient, different intensities of a specific technological parameter) in terms of VOC content. Here, we report the experimental protocols exemplifying different possible applications of our methodology: i.e. the detection of VOCs released during lactic acid fermentation of yogurt (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening), and the in vivo study of retronasal VOC release during coffee drinking (nosespace analysis).

Proton Transfer Reaction Time of Flight Mass Spectrometry allows high-sensitivity, rapid and non-invasive analysis of volatile organic compounds. To demonstrate its potential, we give three examples: lactic acid fermentation of yogurt (on-line bioprocess monitoring), different apple genotypes (large-scale screening), and retronasal space after drinking coffee (nosespace analysis).

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltrimethoxysilane (APTMS) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTMS. The deposition of APTMS and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.

Preparation of Polypentafluorophenyl acrylate Functionalized SiO2 Beads for Protein Purification

A protocol for the preparation of poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads is presented. The poly(PFPA) functionalized surface is then immobilized with antibodies and used successfully for the protein separation through immunoprecipitation.

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

Characterizing Lewis Pairs Using Titration Coupled with In Situ Infrared Spectroscopy

Lewis acid-activation of carbonyl-containing substrates is a fundamental basis for facilitating transformations in organic chemistry. Historically, characterization of these interactions has been limited to models equivalent to stoichiometric reactions. Here, we report a method utilizing in situ infrared spectroscopy to probe the solution interactions between Lewis acids and carbonyls under synthetically relevant conditions. Using this method, we were able to identify 1:1 complexation between GaCl3 and acetone and a highly ligated complex for FeCl3 and acetone. The impact of this technique on mechanistic understanding is illustrated by application to the mechanism of Lewis acid-mediated carbonyl-olefin metathesis in which we were able to observe competitive binding interactions between substrate carbonyl and product carbonyl with the catalyst.

Here, we present a method for the observation of solution interactions between Lewis acids and bases by employing in situ infrared spectroscopy as a detector for titration under synthetically relevant conditions. By examining solution interactions, this method represents a complement to X-ray crystallography, and provides an alternative to NMR spectroscopy.

Lewis acid-activation of carbonyl-containing substrates is a fundamental basis for facilitating transformations in organic chemistry. Historically, ...