We thank Pierre Thibault, Eric Bonneil, Jo&#235;l Lanoix, Caroline C&#244;t&#233; (Institute for Research in Immunology and Cancer, Universit&#233; de Montr&#233;al) and Anthony Purcell (Monash University) for their insightful comments. This work was supported by funding from the Fonds de recherche du Qu&#233;bec - Sant&#233; (FRQS), the Cole Foundation, CHU Sainte-Justine, and the Charles-Bruneau Foundations, Canada Foundation for Innovation, the National Sciences and Engineering Research Council (NSERC) (#RGPIN-2020-05232) and the Canadian Institutes of Health Research (CIHR) (#174924).

The protocol provided here was adapted from established protocols13,14,15,16,17,18,19,20. The overall procedure for the immunoaffinity purification (IP) of MHC class I and II peptides is illustrated in Figure 1. See Table of Materials for details about the cell lines and antibodies used.
1. Beads coupling with the antibodies (Day 1): Coupling antibodies to sepharose CNBr-activated beads
NOTE: Prepare fresh solutions for each new experiment. Refer to Table of Materials and Supplementary Table 1 for the list of reagents and solutions recipes. All the steps are carried out at room temperature (RT). When using a new antibody, check the binding efficiency by collecting key aliquots (see the indication OPTIONAL) and performing Coomassie blue staining SDS-PAGE (Figure 2).
<ol>
	<li>Activation of the sepharose CNBr beads
	<ol>
		<li>Weigh 80 mg of sepharose CNBr-activated beads per sample and transfer them to a 15 mL conical tube.</li>
		<li>To facilitate resuspension of dried beads, first, add 5 mL of 1 mM HCl and pipette up and down 5 times. Then, fill the conical tube with an additional 8.5 mL of 1 mM HCl.</li>
		<li>Rotate at 20 rpm (revolutions per minute) for 30 min at RT using a rotator device. Centrifuge the beads at 200 x g for 2 min at RT and remove the supernatant by aspiration.</li>
		<li>Add 500 &#181;L of coupling buffer to the beads pellet and transfer to a new 2.0 mL centrifuge tube and keep aside for step 1.2.2.</li>
	</ol>
	</li>
	<li>Coupling of antibodies to CNBr-activated beads
	<ol>
		<li>Prepare the antibody selected for the isolation of the MHC class I or II peptides in a new 2.0 mL microcentrifuge tube by adding 2 mg of the antibody (according to the concentration specified by the manufacturer). Complete the volume to 1 mL with the coupling buffer solution to get a final concentration of 2 mg/mL.</li>
		<li>Centrifuge the beads from step 1.1.4 at 200 x g for 2 min at RT and then remove the supernatant by aspiration.</li>
		<li>Add the antibody solution to the 2.0 mL microcentrifuge tube containing the activated beads.</li>
		<li>(OPTIONAL) Take an aliquot of 18 &#181;L (INPUT), add 6 &#181;L of 4x SDS-PAGE buffer, and freeze immediately.</li>
		<li>Rotate the microcentrifuge tube from step 1.2.3 at 20 rpm for 120 min using a rotator device. Centrifuge the beads at 200 x g for 2 min and then remove the supernatant.</li>
		<li>(OPTIONAL) Take an aliquot of 18 &#181;L of the supernatant (unbound antibody), add 6 &#181;L of 4x SDS-PAGE buffer and freeze immediately.</li>
	</ol>
	</li>
	<li>Blocking and washing of antibody coupled-beads
	<ol>
		<li>Add 1 mL of 0.2 M glycine to the microcentrifuge tube containing the antibody-coupled beads from step 1.2.5. Rotate at 20 rpm for 60 min at RT using a rotator device.</li>
		<li>Centrifuge the beads at 200 x g for 2 min, then remove the supernatant. Add 1 mL of PBS (Phosphate-buffered saline).</li>
		<li>Centrifuge the beads at 200 x g for 2 min, then remove the supernatant.</li>
		<li>(OPTIONAL) Take an aliquot of 18 &#181;L of the supernatant (unbound antibody, last wash), add 6 &#181;L of 4x SDS-PAGE buffer and freeze immediately.</li>
		<li>Add 1 mL of PBS to the beads in step 1.3.3.</li>
		<li>(OPTIONAL) Take an aliquot of 18 &#181;L of beads mixture (beads coupled with antibody), add 6 &#181;L of 4x SDS-PAGE buffer, and freeze immediately.</li>
		<li>Keep at 4 &#176;C until use the same day in step 2.5. 
		&#8203;NOTE: Beads can be prepared the day before the immunocapture, but longer storage has not been tested.</li>
	</ol>
	</li>
</ol>
2. Cell lysis and immunocapture with antibody coupled-beads (Day 1)
NOTE: The level of MHC molecules varies from one cell type to another, and the quantification of MHC/HLA molecules per cell is suggested21&#160;(Figure 4A). We recommend a minimum of 1 x 108 cells for each IP. This number of cells corresponds to 6-10 mg of protein from JY, EL4, and A20 cells solubilized with 0.5% Chaps buffer. To prepare cell pellets for IPs, cells should be harvested, centrifuged, and washed twice with 5 mL of PBS. Then, cell pellets can be stored in a 1.5 mL microcentrifuge tube or a 15 mL conical tube at -80 &#176;C until the time of the IP. Note that IPs can be performed on fresh harvested or frozen cell pellets.
<ol>
	<li>To isolate MHC class I or II peptides, thaw a frozen pellet of 1 x 108 cells by warming the bottom of the tube with the palm. Add 500 &#181;L of PBS to the pellet and pipette up and down until the suspension is homogenous. 
	NOTE: Depending on the cell type, the cell pellet volume can vary substantially. If 500 &#181;L of PBS is not enough to dissolve the cell pellet, use more PBS until the cells disaggregate easily while pipetting up and down.</li>
	<li>Measure the total volume of the pellet resuspended in PBS and transfer into a new tube 2 mL microcentrifuge tube. Split into more tubes if needed.</li>
	<li>Add a volume of cell lysis buffer (1% chaps buffer in PBS containing protease inhibitors, 1 pellet/10 mL of buffer) equivalent to the volume of cell pellet resuspended in PBS measured in the previous step. The final concentration of the lysis buffer is 0.5% Chaps.</li>
	<li>Rotate at 10 rpm for 60 min at 4 &#176;C using a rotator device. Centrifuge the cell lysate at 18,000 x g for 20 min at 4 &#176;C with full brake and transfer the supernatant (containing the MHC-peptides complexes) in a new 2.0 mL microcentrifuge tube.</li>
	<li>Recover the antibody-coupled beads from step 1.3.7 by centrifugation at 200 x g for 2 min and remove the supernatant.</li>
	<li>Transfer the cell lysate supernatant in step 2.4 to the antibody-coupled beads and incubate with rotation (10 rpm) for 14-18 h (overnight) at 4 &#176;C using a rotator device.</li>
</ol>
3. Elution of MHC peptides (Day 2)
NOTE: Polypropylene column allows elution of MHC-peptide complexes while retaining the beads into the column. In order to evaluate the proportion of MHC-peptides complexes bounded to the beads before and after acidic elution with 1% TFA (trifluoroacetic acid), it is possible to perform a western blot with aliquots taken at key steps during the protocol (see the indication OPTIONAL). Western blotting shown in Figure 3 reveals the enrichment of MHC-peptide complexes following acidic elution with 1% TFA. The absence of signal in this fraction would indicate that the elution step was not successful. Note that the proportion of unbound MHC-peptides complexes to the beads can also be evaluated in parallel by western blotting using an aliquot described in step 3.1.6.
<ol>
	<li>Elution of MHC-peptide complexes from the antibody coupled-beads
	<ol>
		<li>Remove the bottom lid of the polypropylene column, place the column onto the polypropylene column rack and install an empty container underneath to collect flow through. 
		NOTE: One column per sample is required.</li>
		<li>Rinse the polypropylene column with 10 mL of buffer A and let it drain by gravity. If the flow speed of liquid elution is too slow, further cut the bottom tip of the polypropylene column.</li>
		<li>Measure and collect the beads-lysate mixture (~2 mL) from step 2.5 and transfer it into the polypropylene column.</li>
		<li>(OPTIONAL) Take an aliquot of 20 &#181;L (or 1/100 out of the total volume) for western blotting and freeze immediately. This fraction corresponds to the total MHC-peptides complexes incubated with the beads.</li>
		<li>Let the liquid mixture elute by gravity.</li>
		<li>(OPTIONAL) Collect and measure the flow-through and take an aliquot of 20 &#181;L (or 1/100 out of the total volume) for western blotting and freeze immediately. This fraction represents the residual unbound MHC-peptides complexes.</li>
		<li>To recover the beads-lysate mixture as much as possible, rinse the tube from step 3.1.3 with 1 mL of buffer A and transfer it to the polypropylene column.</li>
		<li>Wash the beads retained in the polypropylene column by adding 10 mL of buffer A. Let the washing buffer elute by gravity.</li>
		<li>Repeat the washing step with 10 mL of buffer B, 10 mL of buffer A and then 10 mL of buffer C.</li>
		<li>Remove the polypropylene column from the rack and place it on the top of a new 2.0 mL microcentrifuge tube. Hold the column and the tube together with the hand.</li>
		<li>Add 300 &#181;L of 1% TFA to the polypropylene column and mix the beads by pipetting up and down 5 times. 
		NOTE: The beads will be retained in the polypropylene column, and the MHC-bound peptides will elute in the 2.0 mL microcentrifuge tube.</li>
		<li>Transfer the eluate in a new 2.0 mL microcentrifuge tube. Repeat step 3.1.11 and pool the 2 eluates (the eluates containing the MHC-peptide complexes should correspond to a total of 600 &#181;L and will be used at step 3.2.4).</li>
		<li>(OPTIONAL) Collect an aliquot of 6 &#181;L (or 1/100 of the total volume) for western blotting and freeze immediately. This fraction corresponds to the MHC-peptides complexes eluted from the beads.</li>
	</ol>
	</li>
	<li>Desalting and elution of MHC peptides 
	NOTE: Desalting and elution of MHC peptides steps could be done by installing the C18 column onto a 2.0 mL microcentrifuge tube. For a better fit, install the anneals provided by the manufacturer between the C18 column and the 2.0 mL microcentrifuge tube. In this protocol, a C18 column is used with a volume capacity of 5-200 &#181;L (6-60 &#181;g). All the steps are carried out at RT.
	<ol>
		<li>Add 200 &#181;L of Methanol on top of the C18 column, then centrifuge at 1546 x g for 3 min. Discard the flow-through.</li>
		<li>Add 200 &#181;L of 80%ACN (acetonitrile)/0.1%TFA on top of the C18 column, then centrifuge at 1546 x g for 3 min. Discard the flow-through.</li>
		<li>Add 200 &#181;L of 0.1%TFA on top of the C18 column, then centrifuge at 1546 x g for 3 min. Discard the flow-through.</li>
		<li>Load 200 &#181;L of the MHC-peptide complexes from step 3.1.12 on top of the C18 column. Centrifuge at 1546 x g for 3 min and discard the flow through.</li>
		<li>Repeat step 3.2.4 twice until the complete volume has been loaded. Note that MHC peptides are retained in the C18 column.</li>
		<li>Add 200 &#181;L of 0.1%TFA to the C18 column, then centrifuge at 1546 x g for 3 min. Discard the flow-through.</li>
		<li>Transfer the C18 column into a new 2.0 mL microcentrifuge tube. Elute MHC peptides from the C18 column by adding 150 &#181;L of 28%ACN/0.1%TFA.</li>
		<li>Centrifuge at 1546 x g for 3 min.</li>
		<li>Transfer the flow-through in a new 1.5 mL microcentrifuge tube. Be careful not to discard the flow-through; it contains the isolated MHC class I or II peptides.</li>
		<li>Repeat steps 3.2.7-3.2.9 twice for a total volume of 450 &#181;L.</li>
		<li>Freeze the 450 &#181;L of eluate (purified MHC class I or II peptides) at -20 &#176;C until samples are analyzed by LC-MSMS.</li>
		<li>Prior to LC-MS/MS analysis, the purified MHC class I or II peptides from step 3.2.11 are evaporated to dryness using a vacuum concentrator with presets of 45 &#176;C for 2 h, vacuum level: 100 mTorr and vacuum ramp: 5. 
		&#8203;NOTE: The evaporation of frozen samples is highly efficient. Dried peptides can be re-frozen until analysis.</li>
	</ol>
	</li>
</ol>
4. Identification of MHC class I and II peptides by LC-MS/MS
NOTE: Analyze the MHC class I and II immunopeptidome using a High-performance Orbitrap and high-resolution quadrupole time-of-flight mass spectrometers6. The following information is given as an indication only, taking into account that the various existing tandem mass spectrometry instruments operate according to different operational standards. A brief outline of the steps are discussed below :
<ol>
	<li>Solubilize the dried samples (from step 3.2.12) in 50 &#181;L of 4% formic acid (FA).</li>
	<li>Load three injections of 16 &#181;L for each sample and separate on a home-made reversed-phase column (150-&#181;m i.d. by 250 mm length, Jupiter 3 &#181;m C18 300 &#197;) with a gradient from 5%-30% ACN-0.1% FA and a 600 nL/min flow rate on a nano-flow UHPLC connected to an MS.</li>
	<li>Acquire each full MS spectrum at a resolution of 120000, an AGC of 4 x 105 with automatic mode for the injection time and spectra using tandem-MS (MS-MS) on the most abundant multiply charged precursor ions for a maximum of 3 s. 
	NOTE: Tandem-MS experiments are performed using higher-energy collisional dissociation (HCD) at a collision energy of 30%, a resolution of 30,000, an AGC of 1.5 x 105, and an injection time of 300 ms.</li>
	<li>Process the data files from the three injections/sample using a proteomics LC-MS/MS analysis software (e.g., PEAKS X) using the mouse and human databases (UniProtKB/Swiss-Prot (2019_09)).</li>
	<li>Select &#39;Unspecified Enzyme Digestion&#39; for the enzyme parameter, and 10 ppm and 0.01 Da for mass tolerances on precursor and fragment ions, respectively. 
	&#8203;NOTE: Variable modifications are deamidation (NQ) and oxidation (M). All other search parameters are the default values. Final peptide lists are filtered using ALC of 80% and with a false discovery rate (FDR) of 1 % using the proteomics LC-MS/MS analysis software.</li>
</ol>
5. Visualization of immunopeptidomics data
NOTE: The quality of immunopeptidomics data generated by MS can be assessed in multiple ways, as recently described22,23. To visualize the data and assess their overall quality, composition, and MHC-specificity, the MhcVizPipe (MVP) software tool can be used.
<ol>
	<li>Follow all the instructions and related documentation to install and run the MVP software available at the Caron Lab GitHub website24. 
	NOTE: MVP provides a rapid and consolidated view of sample quality, composition, and MHC-specificity. MVP parallelizes the use of well-established immunopeptidomics algorithms (NetMHCpan25, NetMHCIIpan26, and GibbsCluster27) and generates organized and easy-to-understand reports in HTML (HyperText Markup Language) format. The reports are fully portable and can be viewed on any computer with a modern web browser. See Supplementary Data 1-4 for examples of HTML reports.</li>
</ol>

The authors declare that they have no competing financial interests.

Two mouse cell lines (EL4 and A20), one human cell line (JY), and five commercially available antibodies [M1 (anti-H2Db/Kb), Y3 (anti-H2Kb), M5 (anti-H2-IAd/IEd), W6/32 (anti-HLA-ABC), and L243 (anti-HLA-DR)] were tested and validated in the context of this protocol and provide high-quality immunopeptidomics data. Other anti-HLA antibodies are available (e.g., anti-HLA-A2 BB7.2) but were not tested here. Note that the W6/32 antibody is widely used and the most established antibody in the field; it enables the isolation of peptides presented by all HLA-ABC molecules in humans and was previously reported by expert laboratories to work from various biological sources such as fresh or frozen tissues8,39, peripheral blood mononuclear cells and bone marrow mononuclear cell40, biopsies41, xenografts41,42, autopsies43 and plasma samples44,45.
The preparation of fresh solutions that are used throughout the protocol is critical. In particular, the use of fresh acidic solutions in glass bottles is critical to avoid subsequent contamination of the samples analyzed by MS. In addition, when the protocol is done for the first time and/or with a new antibody, it is important to assess that the antibody is indeed coupled to the CNBr Sepharose beads using a blue Coomassie gel. The washing steps of the antibody coupled beads following immunocapture of the MHC-peptide complexes is also critical to avoid contamination of non-MHC peptides. Finally, special care is required to not discard the eluates following the elution of the MHC-peptide complexes with 1% TFA and the elution of the peptides from the C18 column with ACN28%/0.1% FA.
Existing protocols available in the literature describe additional steps to further purify the peptides at the end of the isolation procedure, e.g., peptide fractionation by different methods14,46 or ultrafiltration using 10-30 kDa filters13,47. The current protocol does not provide details on those additional steps and is sufficient to provide high-quality immunopeptidomics data. However, such steps could be considered by non-experts to modify and troubleshoot to further optimize the peptide isolation procedure.
The type of beads and the type of acidic elution buffer that are used to elute MHC-complexes from the beads can also be modified for troubleshooting13,14,15,16,17,18,19. In this regard, sepharose CNBr-activated beads are generally a good starting point since they are relatively inexpensive in addition to show flexibility in terms of binding with various types of antibodies. In the current protocol, sepharose CNBr-activated beads were shown to perform relatively well using five different commercially available antibodies (i.e., M1, Y3, W6/32, L243, and M5). Besides sepharose CNBr-activated beads, Protein A or G or A/G sepharose 4 Fast Flow beads are also easy to handle, and although relatively more expensive, can generate similar results. Another factor to consider is the affinity of the antibody for protein A or G. Furthermore, sepharose magnetic beads are also very easy to use but are relatively expensive. Independently of the type of beads selected by non-experts, it is encouraged to collect aliquots at critical steps of the protocol and perform blue Coomassie-stained SDS-PAGE gels to track the binding efficiency of the antibody to the beads, as shown in Figure 2.
Another important factor influencing the successfulness of MHC peptides isolation refers to the type of acidic elution buffer used to isolate the MHC-peptide complexes from the beads. Different buffers have been reported including 0.1%, 1% or 10% TFA, 0.2% FA and 10% acetic acid. 1% TFA was the elution buffer working for all the antibodies tested. This step could also be traced by western blotting against the MHC molecules used to capture MHC peptides, as shown in Figure 3.
All buffers containing acetonitrile (ACN) and/or trifluoroacetic acid (TFA) are aggressive and can lead to contamination of the sample with small molecular and polymeric substances such as plasticizers if in contact with plastic. To avoid such issues, all solutions containing an organic solvent and/or TFA are prepared fresh daily and stored in a glass bottle until use. The majority of the steps are performed in Protein LoBind plastic tube. These tubes are specifically designed for proteomics and are made of the highest quality, virgin polypropylene free of biocides, plasticizers, and latex. They are also produced with optimized, highly polished molds without the use of slip agents. These precautions are important to consider to enable the generation of high-quality immunopeptidomics data.
The antibody is an important limitation for the isolation of MHC-bound peptides. The W6/32 antibody enables the isolation of peptides presented by all HLA-ABC class I molecules in humans and is the most widely used and established antibody in the field. High-resolution HLA typing of uncharacterized human cell lines or biospecimens is not a necessity upon application of the W6/32 antibody but is nevertheless recommended for certain applications to facilitate data interpretation48. HLA/MHC typing information can also be found in public resources for multiple cell lines and mouse models49. Besides the W6/32 antibody, the four other antibodies (M1, M5, Y3, and L243) that were tested and validated in the context of this protocol are all commercially available. On the other hand, many other antibodies that were reported in previous immunopeptidomics studies have not been largely adopted by the community and are not commercially available or are available through the culture of hybridoma cell lines, which is relatively expensive.
Another important limitation for the isolation of MHC-bound peptides is the amount of starting material required. The amount required is inversely proportional to the expression level of MHC molecules on the cell surface, which can be quantified by flow cytometry (Figure 4A). Cells showing high expression levels of MHC molecules (e.g., dendritic cells and hematopoietic cells in general) generally yield high-quality immunopeptidomics data. Expert labs use from as low as 50 million cells50, but 100 million to 1 billion cells are recommended for non-experts. Use of tissue biopsy (&#60;13 mg)41, xenograft42,43, autopsy44, and plasma45,46 samples were also reported but remain challenging for non-expert laboratories. Also, the total number of MHC-associated peptides expected is well documented for established cell lines (here, between ~2000 and ~10000 peptides depending on the cell line and antibody used), but the absolute amounts of naturally presented peptides that are efficiently pulled down by the technique remain debated. Indeed, previous studies estimated that the efficiency of the isolation procedure is peptide-dependent and can be from as low as 0.5%-2%51. Other limitations in immunopeptidomics are the reproducibility of the methods and the inability of the NetMHCpan suite tools to correctly annotate peptides to MHC alleles that are less characterized. In this regard, further development and application of relatively new data-independent acquisition MS methods7,32,52,&#160;as well as new peptide clustering and MHC peptide binding prediction algorithms31,34,53,54 are expected to improve the reproducibility and the accuracy of peptide annotation in immunopeptidomics. Immunopeptidomics is facing other limitations regarding MS acquisition and computational analysis of MHC-associated peptides and are covered elsewhere1,6,55.
While the isolation of HLA-ABC-associated peptides from human samples using the W6/32 antibody is well established and widely applied by many research groups, the isolation of mouse MHC class I- and II-associated peptides is relatively less established. Hence, robust protocols for the isolation of mouse MHC ligands are needed. Here, we provide a protocol optimized for the isolation of MHC class I peptides and MHC class II peptides from two mouse cell lines of C57BL/6 and BALB/c origin, respectively. Specifically, the protocol enables the isolation of class I H2Kb- and H2Db-associated peptides using the M1 antibody, as well as class II H2-IAd and H2-IEd-associated peptides using the M5 antibody. Thus, dissemination and application of the current protocol should facilitate basic and translational immunopeptidomics research in various mouse models.
The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. For instance, it can be adapted and further optimized to perform immunopeptidomics screening in various biological matrices ranging from blood/plasma to fresh or frozen tissue to FFPE (Formalin-Fixed Paraffin-Embedded). Moreover, the protocol will foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics and is therefore expected to find wide application in basic and clinical research.

Over the last decade, the interest in investigating the repertoire of MHC-associated peptides has exceeded the academic sector and reached the biotech and pharmaceutical industries. Indeed, in cancer, the discovery of actionable tumor-specific neoantigens represents a major research focus in the industrial sector to develop clinical immunotherapies leading to personalized oncology1,2,3. Fundamentally, MHC-associated peptides are presented throughout the entire body, reflect the intracellular stage of the cell, and are significant in various disease conditions such as autoimmunity, transplantation, infectious diseases, inflammation, cancer, and allergies1,4. Thus, MHC-associated peptides, or human leukocyte antigen (HLA) ligands in humans, are of great medical interest and are collectively referred to as the immunopeptidome5.
MS is a powerful analytical approach to characterize the immunopeptidome6,7, including the discovery of tumor-specific neoantigens8,9,10,11. A typical workflow to perform an immunopeptidomics experiment includes three main steps: 1) sample preparation for the isolation of MHC-associated peptides, 2) data acquisition by MS, and 3) data analysis using various computational software tools12. The generation of high-quality samples described in this visualized protocol is critical for the success of any project in MS-based immunopeptidomics. The protocol described below focuses on isolating MHC class I- and II-associated peptides from well-established cell lines that are suitable for generating high-quality immunopeptidomics data. Representative results from those cell lines are shown in the current protocol.

<table><tbody><tr><td>A20 cell line</td><td>ATCC</td><td>TIB-208</td><td>mouse B lymphoblast</td></tr><tr><td>Acetonitrile, LC/MS Grade</td><td>FisherScientific</td><td>A955-4</td><td></td></tr><tr><td>anti-Human HLA A, B, C (W6/32) - MHC class I</td><td>BioXcell</td><td>BE0079</td><td></td></tr><tr><td>anti-Human/Monkey HLA-DR (L243) - MHC class II</td><td>BioXcell</td><td>BE0308</td><td></td></tr><tr><td>anti-Mouse H2 (M1/42.3.9.8) - MHC class I</td><td>BioXcell</td><td>BE0077</td><td></td></tr><tr><td>anti-Mouse H2-IAd/IEd (M5/114) - MHC class II</td><td>BioXcell</td><td>BE00108</td><td></td></tr><tr><td>anti-Mouse H2Kb (Y3) - MHC class I</td><td>BioXcell</td><td>BE0172</td><td></td></tr><tr><td>BupH Phosphate Buffered Saline Packs (PBS)</td><td>ThermoFisher</td><td>28372</td><td>Pouch contents dissolved in a final volume of 500 mL deionized water (FisherScientific, W64)</td></tr><tr><td>CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)</td><td>EMDMilipore</td><td>220201-10MG</td><td></td></tr><tr><td>CNBr-activated Sepharose</td><td>Cytivia</td><td># 17-0430-01</td><td></td></tr><tr><td>EL4 cell line</td><td>ATCC</td><td>TIB-39</td><td>mouse T lymphoblast</td></tr><tr><td>epTIPS LoRetention Tips, 1000 &amp;micro;L/Eppendorf</td><td>FisherScientific</td><td>02-717-352</td><td>Better results with low retention material</td></tr><tr><td>epTIPS LoRetention Tips, 200 &amp;micro;L/Eppendorf</td><td>FisherScientific</td><td>02-717-351</td><td>Better results with low retention material</td></tr><tr><td>Formic Acid, LC/MS Grade</td><td>FisherScientific</td><td>A117-50</td><td></td></tr><tr><td>Glycine</td><td>FisherScientific</td><td>RDCG0250500</td><td></td></tr><tr><td>Hydrochloric acid solution</td><td>FisherScientific</td><td>60-007-11</td><td></td></tr><tr><td>JY cell line</td><td>Sigma Aldrich</td><td>94022533-1VL</td><td>EBV-immortalised B cell lymphoblastoid line</td></tr><tr><td>Methanol, LC/MS Grade</td><td>FisherScientific</td><td>A456-4</td><td></td></tr><tr><td>Poly prep chromatography columns (polypropylene column)</td><td>Bio-Rad</td><td>731-1550</td><td>referred as polypropylene column in the protocol</td></tr><tr><td>Proteases inhibitor</td><td>ThermoFisher</td><td>A32963</td><td>1 pellet per 10 mL of cell lysis buffer</td></tr><tr><td>Qifikit</td><td>Dako</td><td>K007811-8</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Amresco</td><td># 0865-1kg</td><td></td></tr><tr><td>Sodium Chloride</td><td>FisherScientific</td><td>MSX04201</td><td></td></tr><tr><td>Solid phase extraction disk, ultramicrospin column C18</td><td>The nest group</td><td>SEMSS18V</td><td>capacity of 6&amp;ndash;60 &amp;micro;g, max volume of 200 &amp;micro;L</td></tr><tr><td>Trifluoroacetic Acid (TFA), LC-MS Grade</td><td>FisherScientific</td><td>PI85183</td><td></td></tr><tr><td>Tris</td><td>FisherScientific</td><td>T395-500</td><td></td></tr><tr><td>Tris-HCl</td><td>FisherScientific</td><td>#10812846001</td><td></td></tr><tr><td>Tube LoBind 1.5 mL/Eppendorf</td><td>FisherScientific</td><td>E925000090</td><td>Better results with low retention material</td></tr><tr><td>Tube LoBind 2 mL/Eppendorf</td><td>FisherScientific</td><td>13-698-795</td><td>Better results with low retention material</td></tr><tr><td>Water, LC/MS Grade</td><td>FisherScientific</td><td>W64</td><td></td></tr></tbody></table>

immunopeptidomics: isolation of mouse and human mhc class i- and ii-associated peptides for mass spectrometry analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

The general workflow to isolate MHC-peptide complexes for the analysis of immunopeptidomes by MS is illustrated in Figure 1. Representative results for the verification of the beads-binding efficiency of the antibody (Figure 2) (using the Y3 anti-H2Kb antibody) and the elution efficiency of the MHC-peptide complexes from the beads (Figure 3) (using the W6/32 anti-HLA-ABC antibody) are shown. Flow cytometry-based quantification assays21 were also applied to measure the absolute number of MHC class I molecules per EL4 cell (H2Kb and H2Db) and JY cell (HLA-ABC), as shown in Figure 4A.
The intra- and inter-individual reproducibility of the results using the current protocol are shown in Figure 4B-E. Representative results are shown for MHC class I peptides identified from 1 x 108 EL4 cells and 1 x 108 JY cells. Results were generated from two different lab members (User 1 and User 2). For User 1, the mean number of MHCI-specific peptides detected from EL4 and JY cells was 1341 and 6361, respectively; for User 2, 1933 and 7238, respectively (Figure 4B,D). The average coefficient of variation (CV) for the number of peptides detected across three different biological replicates/experiments varies from 1.9%-11% (Figure 4B,D). Although the CVs for the number of peptides detected across the three different experiments were relatively small, the identity of the peptides varied considerably (Figure 4C,E). Indeed, representative Venn diagrams show that the proportion of peptides that were reproducibly detected across three biological replicates ranged from 39% (User 2, EL4 cells) to 63% (User 1, JY cells) (Figure 4C,E and Supplementary Table 2).
Heatmaps generated by the MVP software show predicted MHC binding strength of the identified peptides using the NetMHCpan suite tools25,26,28. Two GibbsCluster routine options, which are termed 'Unsupervised GibbsCluster' and 'Allele-Specific GibbsCluster', are also performed by MVP to extract MHC peptide-binding motifs. Note that MVP has limitations; its primary goal is not to extract allele-specific motifs and annotate peptides in a highly accurate manner but to rather provide a bird's eye view on the overall quality, composition, and MHC-specificity of the samples.
For mouse MHC class I (H2Db and H2Kb) peptides in EL4 cells (Figure 5A; upper panels and Supplementary Data 1), a representative heatmap shows that 89% of all detected 8-12-mer peptides are predicted to be Strong Binders (SB: NetMHCpan %Rank &lt;0.5) or Weak Binders (WB: NetMHCpan %Rank &lt;2) for H2Db or H2Kb molecules. Sequence clusters generated from the 8-12-mer peptides are in concordance with reported logos for H2Db (asparagine at P5 and Leucine at P9) and H2Kb (phenylalanine at P5 and leucine at P8) (Figure 5)29. It is worth mentioning that a third dominant motif (histidine at P7 and leucine at P9) as well as additional 'artefactual' motifs can be observed using the M1 antibody (Supplementary Data 1). In fact, the M1 antibody is known to cross-react with the non-classical Qa2 molecule, and therefore, Qa2-associated peptides are also detected by MS (Supplementary Data 1). Here, for simplicity, Figure 5 focuses on showing the well-established peptide binding motifs for the two classical H2b alleles (i.e., H2Db or H2Kb) expressed in EL4 cells.
For human HLA class I (HLA-ABC) peptides in JY cells (Figure 5A; lower panels and Supplementary Data 2), a representative heatmap shows that 97% of all detected 8-12-mer peptides are predicted to be SB or WB for HLA-A*0201, -B*0702 or -C*0702. Peptides were clustered to visualize peptide binding motifs for HLA-A*0201 and -B*0702. Binding motif for HLA-C*0702 is not shown in Figure 5A because the C*0702 allele has a relatively low expression level. Therefore too few C*0702 peptides were isolated and identified to generate a representative C*0702 motif.Note that the C*0702 motif can be visualized in other studies30,31,32 or from the NetMHCpan 4.1 Motif Viewer website33.
For human HLA class II (HLA-DR) peptides in JY cells (Figure 5B; upper panels and Supplementary Data S3), a representative heatmap shows that 70% of all detected 9-22-mer peptides are predicted to be SB or WB for HLA-DRB1*0404 and -DRB1*1301. Peptide binding motifs for these two alleles are shown (Figure 5B). Note that the peptide-binding motifs shown here may not be in complete concordance with the recently reported logos for HLA-DRB1*0404 and -DRB1*130134. This discrepancy highlights the current inability of MVP/NetMHCpan to precisely annotate peptides to HLA class II alleles that are less characterized, such as HLA-DRB1*0404 and -DRB1*1301 expressed in JY cells. Additional information on class II peptide binding motifs can be found in other studies34,35 and from the NetMHCIIpan 4.0 Motif Viewer website36.
Finally, for mouse MHC class II (H2-IAd and H2-IEd) peptides in A20 cells (Figure 5B; lower panels and Supplementary Data 4), a representative heatmap shows that 87% of all detected 9-22-mer peptides are predicted to be SB or WB for H2-IAd or H2-IEd. Peptide binding motifs for these two alleles are in concordance with reported logos37.
Complete HTML reports generated by the MVP software to assess the overall quality and MHC-specificity of the samples are available in Supplementary Data 1-4.
<img alt="MHC peptide processing diagram: cell lysis, immunocapture, LC-MS/MS data analysis." class="xfigimg" src="/files/ftp_upload/63052/63052fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Schematic of the complete procedure for the isolation of MHC class I and II peptides. (A-B)100 million cells are pelleted and lysed with 0.5% Chaps buffer. (C) The cell lysate is centrifuged, and the supernatant is added to CNBr-sepharose beads coupled to the desired antibody beforehand and (D) incubated 14-18 h at 4 °C. (E) Following immunocapture, the beads are transferred into a polypropylene column and washed, and the MHC-peptide complexes are eluted with a 1% TFA solution. (F) The peptides are desalted and eluted using a C18 column. (G) Subsequently, peptides are speed vac dried and analyzed by tandem mass spectrometry. (H) The quality of the isolated MHC class I and II peptides can be assessed based on the HLA subtypes using the freely available MhcVizPipe software. Figure created with BioRender.com (NT22ZL8QSL). <a href="https://www.jove.com/files/ftp_upload/63052/63052fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="SDS-PAGE diagram showing protein separation using anti-H2Kb antibody; analysis of heavy and light chains." class="xfigimg" src="/files/ftp_upload/63052/63052fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Coomassie gel staining to track antibody binding efficiency to sepharose CNBr-activated beads. Aliquots of equivalent volumes were loaded onto 12% SDS-PAGE gel followed by Coomassie blue staining: beads + antibody pre-coupling (1), unbound antibody following coupling step (2), supernatant following last wash after coupling (3), and beads coupled with antibody (4). The efficiency of binding is illustrated by a significant decrease in signal staining intensity of the light and heavy chains of H2Kb when beads are covalently bound (Lane 4) to the CNBr beads compared to the antibody before coupling (Lane 1). The figure has been reprinted and adapted from bioRxiv38. <a href="https://www.jove.com/files/ftp_upload/63052/63052fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Western blot of MHC-peptide complexes using Anti-HLA-ABC showing protein bands at 33-44 kDa." class="xfigimg" src="/files/ftp_upload/63052/63052fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Western blotting for tracking MHC-peptide complexes following acidic elution from antibody-coupled CNBr-activated beads. Aliquots taken from the steps indicated in the protocol (1/100 of the total measured volume) were loaded onto a 12% SDS-PAGE gel and transferred onto nitrocellulose membrane: Beads + lysate following immunocapture and last wash (A); supernatant of last wash (B) and MHC-peptide complexes eluted from the beads (C). The strong detection signal of the MHC-peptide complexes in (C) using anti-HLA-ABC heavy chain antibody (Abcam, #ab 70328, 1:5000) confirmed the isolation of MHC-peptide complexes following acidic elution. Note that it is possible to assess the proportion of MHC-peptide complexes that were not captured by the antibody coupled beads by collecting the flow-through from step 3.1.6. An aliquot can be added to the western blot (not shown on this gel). <a href="https://www.jove.com/files/ftp_upload/63052/63052fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Protein ligand quantification charts; bar graphs, Venn diagrams; human vs mouse cell data analysis." class="xfigimg" src="/files/ftp_upload/63052/63052fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Identification of MHC class I peptides from JY and EL4 cells. (A) Histogram showing the absolute number of cell surface H2Db and H2Kb molecules per EL4 cell and of HLA-ABC molecules per JY cell. Quantification was performed by flow cytometry. Mean and standard error of the mean were obtained from three biological replicates. (B, D) Histogram showing the mean number and standard deviation of the mean of MHC class I peptides identified by two independent users (USER_1 [U1] and USER_2 [U2]). Mean number and standard deviation of the mean of MHC class I peptides detected from mouse EL4 cells (B) and human JY cells (D) are shown. Coefficients of variation (CV) across three independent biological replicates are indicated. (C, E) Venn diagrams showing the number of peptides that were reproducibly detected in EL4 (C) and JY cells (E) by two independent users (U1 and U2) across three independent biological replicates. Figure 4A has been reprinted and adapted from bioRxiv38. <a href="https://www.jove.com/files/ftp_upload/63052/63052fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="MHC class I and II peptide binding motifs; HLA and H-2 alleles; sequence logo diagrams; immune analysis." class="xfigimg" src="/files/ftp_upload/63052/63052fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Visualization of immunopeptidomics data using the MVP software tool. Data analysis of mouse and human (A) MHC class I and (B) MHC class II peptides. Representative binding affinity heatmaps (left panels) and peptide binding motifs (right panels) are shown. Heatmaps colors represent the MHC binding affinity predicted by NetMHCpan 4.1 (%rank). Strong Binders are red (%rank &lt;0.5), Weak Binders are blue (%rank &lt;2) and Non-Binders are yellow (%Rank &gt;2). Proportion (%) of 8-12mer peptides (A) and 9-22mer peptides (B) that are SB or WB is indicated in parenthesis. Peptide binding motifs were generated by MVP using the 'Allele-specific Gibbscluster' option. These representative results were obtained from 1 x 108 cells and by using the following antibodies and cell lines: M1 antibody and EL4 cells for mouse MHC class I peptides; W6/32 antibody and JY cells for human MHC class I peptides; M5 antibody and A20 cells for mouse MHC class II peptides; L243 antibody and JY cells for human MHC class II peptides. Refer to the Supplementary Data 1-4 to access the full HTML reports generated by the MVP software. <a href="https://www.jove.com/files/ftp_upload/63052/63052fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Data 1-4: <a href="https://www.jove.com/files/ftp_upload/63052/SUPPLEMENTARY%20DATA%20S1-S4.zip" target="_blank">Please click here to download this File.</a>
Supplementary Data 1: HTML report generated by the MVP software from peptides that were isolated from 1 x 108 EL4 cells using the M1 antibody. Three biological replicates are shown. This report is related to Figure 4 and Figure 5 and representative peptides are listed in Supplementary Table 2.
Supplementary Data 2: HTML report generated by the MVP software from peptides that were isolated from 1 x 108 JY cells using the W6/32 antibody. Three biological replates are shown. This report is related to Figure 4, and Figure 5, and representative peptides are listed in Supplementary Table 2.
Supplementary Data 3: HTML report generated by the MVP software from peptides that were isolated from 1 x 108 JY cells using the L243 antibody. This report is related to Figure 5, and representative peptides are listed in Supplementary Table 2.
Supplementary Data 4: HTML report generated by the MVP software from peptides that were isolated from 1 x 108 A20 cells using the M5 antibody. This report is related to Figure 5, and representative peptides are listed in Supplementary Table 2.
Supplementary Table 1: List of buffers. Recipes for all the buffers used in the protocol are described. <a href="https://www.jove.com/files/ftp_upload/63052/List%20of%20Buffers-63052R1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 2: Representative lists of peptides associated with H2Db/Kb, HLA-ABC, HLA-DR, and H2-IAd/IEd. This table contains the lists of representative MHC class I and II peptides isolated from mouse EL4 and A20 cell lines, respectively, and HLA class I and II from human JY cell line. These data have been deposited on the ProteomeXchange (PXD028633). <a href="https://www.jove.com/files/ftp_upload/63052/SUPPLEMENTARY%20TABLE%20S2-R2.xlsx" target="_blank">Please click here to download this Table.</a>
DATA AVAILABILITY:
Datasets used in this manuscript were deposited on the ProteomeXchange (http://proteomecentral.proteomexchange.org/cgi/GetDataset):  PXD028633.

Watch this Scientific Journal Video about Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis at JoVE.com

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

immunopeptidomics-isolation-mouse-human-mhc-class-i-ii-associated

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Biochemistry

11.6K Views.  CHU Sainte-Justine Research Center. Dit protocol is belangrijk omdat het eenvoudig uit te voeren is en binnen het bereik van elk laboratorium ligt. Dit protocol maakt de isolatie van HLA of MHC type I of type II peptiden uit menselijke of muismonsters mogelijk. Deze methode kan wetenschappelijke vragen beantwoorden met betrekking tot alle onderzoeksgebieden met betrekking tot het immuunsysteem, of het nu gaat om kanker, virologie of auto-immuunziekten.Dit protocol is gebouwd om toegankelijk te zijn voor iedereen die in e...

Video: Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

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 ...

Chemistry

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

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Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Enrichment of Host Cell Proteins in Monoclonal Antibody Drug Products

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.

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

Enhancing EV Isolation for Precise Biofluid Analysis

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

Author Spotlight: Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery 

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...

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

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step.
Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers.
Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution.
Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

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