This study is supported by the NIH grant (R01NS121608). A.M.F. acknowledges the ARCS-Metro Washington Chapter Scholarship and the Bourbon F. Scribner Endowment Fellowship. We thank the Michael Ward lab at the National Institute for Neurological Disorders and Stroke (NINDS) for molecular biology support and the i3Neuron technology development.

<ol>
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The authors declare no competing financial interests.

Using this LAMP1-APEX probe, proteins on and near the lysosomal membrane are biotinylated and enriched. Given the typical lysosome diameter of 100-1,200 nm, this method provides excellent intracellular resolution with a 10-20 nm labeling radius. LAMP1 is an abundant lysosomal membrane protein and a classical marker for lysosomes, serving as an excellent bait protein for lysosomal APEX labeling at the endogenous expression level. However, limitations also exist when using LAMP1 to target lysosomes, as LAMP1 is also presen...

Lysosomes are catabolic organelles that degrade macromolecules via the lysosomal-autophagy pathway1. Besides degradation, lysosomes are involved in diverse cellular functions such as signaling transduction, nutrient sensing, and secretion2,3,4. Perturbations in lysosomal function have been implicated in lysosomal storage disorders, cancer, aging, and neurodegeneration3,5,6,7. For postmitotic and highly polarized neurons, lysosomes play critical roles in neuronal cellular homeostasis, neurotransmitter release, and long-distance transport along the axons8,9,10,11. However, investigating lysosomes in human neurons has been a challenging task. Recent advancements in induced pluripotent stem cell (iPSC)-derived neuron technologies have enabled the culture of live human neurons that were previously inaccessible, bridging the gap between animal models and human patients to study the human brain12,13. Particularly, the advanced i3Neuron technology stably integrates the neurogenin-2 transcription factor into the iPSC genome under a doxycycline-inducible promoter, driving iPSCs to differentiate into pure cortical neurons in 2 weeks14,15.
Due to the highly dynamic lysosomal activity, capturing lysosomal interactions with other cellular components is technically challenging, particularly in a high-throughput fashion. Proximity labeling technology is well-suited to studying these dynamic interactions because of its capability to capture both stable and transient/weak protein interactions with exceptional spatial specificity16,17. Engineered peroxidase or biotin ligase can be genetically fused to the bait protein. Upon activation, highly reactive biotin radicals are produced to covalently label neighboring proteins, which can then be enriched by streptavidin-coated beads for downstream bottom-up proteomics via liquid chromatography-mass spectrometry (LC-MS) platforms17,18,19,20,21.
An endogenous lysosomal proximity labeling proteomics method was recently developed to capture the dynamic lysosomal microenvironment in i3Neurons22. Engineered ascorbate peroxidase (APEX2) was knocked-in on the C-terminus of the lysosomal associated membrane protein 1 (LAMP1) in iPSCs, which can then be differentiated into cortical neurons. LAMP1 is an abundant lysosomal membrane protein and a classical lysosomal marker23. LAMP1 is also expressed in late endosomes, which mature into lysosomes; these late endosome-lysosomes and nondegradative lysosomes are all referred to as lysosomes in this protocol. This endogenous LAMP1-APEX probe, expressed at the physiological level, can reduce LAMP1 mislocalization and overexpression artifacts. Hundreds of lysosomal membrane proteins and lysosomal interactors can be identified and quantified with excellent spatial resolution in live human neurons.
Here, a detailed protocol for lysosome proximity labeling proteomics in human iPSC-derived neurons is described with further improvements from the recently published method22. The overall workflow is illustrated in Figure 1. The protocol includes hiPSC-derived neuron culture, proximity labeling activation in neurons, validation of APEX activity by fluorescence microscopy, determination of an optimal streptavidin beads-to-input protein ratio, enrichment of biotinylated proteins, on-beads protein digestion, peptide desalting and quantification, LC-MS analysis, and proteomics data analysis. Troubleshooting guidelines and experimental optimizations are also discussed to improve proximity labeling quality control and performance.

<table><tbody><tr><td>10% (w/v) Saponin solution</td><td>Acros Organics</td><td>419231000</td><td>Flourescent Microscopy</td></tr><tr><td>Accutase</td><td>Life Technologies</td><td>A1110501</td><td>cell detachment solution, Cell Culture</td></tr><tr><td>B27 Supplement</td><td>Fisher Scientific</td><td>17504044</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>BDNF</td><td>PeproTech</td><td>450-02</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B6768</td><td>Cell Culture, Borate Buffer</td></tr><tr><td>Bovine Serum Albumin</td><td>Millipore Sigma</td><td>A8806</td><td>To make standard solutions to measure total protein concentrations</td></tr><tr><td>Brainphys neuronal medium</td><td>STEMCELL Technologies</td><td>5790</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>CD45R (B220) Antibody Alexa Fluor 561</td><td>Thermo Fisher Scientific</td><td>505-0452-82</td><td>Flourescent Microscopy</td></tr><tr><td>Chroman1 ROCK inhibitor</td><td>Tocris</td><td>716310</td><td>Cell Culture</td></tr><tr><td>cOmplete mini Protease Inhibitor</td><td>Roche</td><td>4693123001</td><td>cocktail inhibitor in Lysis Buffer</td></tr><tr><td>DC Protein Assay Kit II</td><td>Bio-Rad</td><td>5000112</td><td>To determine total protein concentrations of cell lysate</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td>Proximity-labeling Reaction</td></tr><tr><td>DMEM/F12 medium</td><td>Thermo Fisher Scientific</td><td>11320082</td><td>Cell Culture, Dish Coating</td></tr><tr><td>DMEM/F12 medium with HEPES</td><td>Thermo Fisher Scientific</td><td>11330057</td><td>Cell Culture, Induction Medium</td></tr><tr><td>Donkey serum</td><td>Sigma-Aldrich</td><td>D9663</td><td>Flourescent Microscopy</td></tr><tr><td>Doxycycline hyclate, &amp;ge;98% (HPLC)</td><td>Sigma-Aldrich</td><td>D9891-1G</td><td>Cell Culture, Induction Medium</td></tr><tr><td>Essential 8 Medium</td><td>Thermo Fisher Scientific</td><td>A1517001</td><td>Cell Culture</td></tr><tr><td>Essential 8 Supplement (50x)</td><td>Thermo Fisher Scientific</td><td>A1517101</td><td>Cell Culture</td></tr><tr><td>Extraction plate vacuum manifold kit</td><td>Waters</td><td>WAT097944</td><td>For Peptide desalting</td></tr><tr><td>Formic Acid (FA)</td><td>Fisher Scientific</td><td>A11750</td><td>For LC-MS analysis</td></tr><tr><td>GDNF</td><td>PeproTech</td><td>450-10</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>Hoechst dye</td><td>Thermo Fisher Scientific</td><td>62239</td><td>Flourescent Microscopy</td></tr><tr><td>HPLC grade methanol</td><td>Fisher Scientific</td><td>A452</td><td>For Peptide desalting</td></tr><tr><td>HPLC grade water</td><td>Fisher Scientific</td><td>W5</td><td>For Peptide desalting</td></tr><tr><td>Human induced pluripotent stem cells</td><td>Corriell Institute</td><td>GM25256</td><td>Cell Culture</td></tr><tr><td>Hydrogen peroxide, ACS, 29-32% w/w aq. soln., stab.</td><td>Thermo Fisher Scientific</td><td>AA33323AD</td><td>Proximity-labeling Reaction</td></tr><tr><td>Iodoacetamide (IAA)</td><td>Millipore Sigma</td><td>I6125</td><td>For Protein Digestion</td></tr><tr><td>Laminin</td><td>Fisher Scientific</td><td>23017015</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>LC-MS grade Acetonitrile</td><td>Fisher Scientific</td><td>A955</td><td>For LC-MS analysis</td></tr><tr><td>LC-MS grade water</td><td>Fisher Scientific</td><td>W64</td><td>For LC-MS analysis</td></tr><tr><td>L-glutamine</td><td>Fisher Scientific</td><td>25-030-081</td><td>Cell Culture, Induction Medium</td></tr><tr><td>Matrigel</td><td>Thermo Fisher Scientific</td><td>08-774-552</td><td>basement membrane matrix, Cell Culture, Dish Coating</td></tr><tr><td>Mouse anti-human LAMP1 monoclonal antibody</td><td>Developmental Studies Hybridoma Bank</td><td>h4a3</td><td>Flourescent Microscopy</td></tr><tr><td>N-2 Supplement (100x)</td><td>Fisher Scientific</td><td>17-502-048</td><td>Cell Culture, Induction Medium</td></tr><tr><td>Nitrocellulose Membrane, Precut, 0.45 &amp;micro;m, 7 x 8.5 cm</td><td>Bio-Rad</td><td>1620145</td><td>To conduct dot blot assay for bead titration</td></tr><tr><td>Non-essential amino acids (NEAA)</td><td>Fisher Scientific</td><td>11-140-050</td><td>Cell Culture, Induction Medium</td></tr><tr><td>NT-3</td><td>PeproTech</td><td>450-03</td><td>Cell Culture, Cortical Neuron Medium</td></tr><tr><td>Oasis HLB 96-well solid phase extraction plate</td><td>Waters</td><td>186000309</td><td>For Peptide desalting</td></tr><tr><td>Odyssey Blocking Buffer (TBS)</td><td>LI-COR Biosciences</td><td>927-50000</td><td>To conduct dot blot assay for bead titration</td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Flourescent Microscopy</td></tr><tr><td>Phenol Biotin (1,000x stock)</td><td>Adipogen</td><td>41994-02-9</td><td>Proximity-labeling Reaction</td></tr><tr><td>Phosphate-buffered saline (PBS) without calcium or magnesium</td><td>Gibco</td><td>10010049</td><td>Cell Culture, Proximity-labeling Reaction, Flourescent Microscopy</td></tr><tr><td>Pierce Quantitative Colorimetric Peptide Assay</td><td>Thermo Fisher</td><td>23275</td><td>Peptide Concentration Assay</td></tr><tr><td>Poly-L-Ornithine (PLO)</td><td>Millipore Sigma</td><td>P3655</td><td>Cell Culture, Dish Coating</td></tr><tr><td>Sodium Ascorbate</td><td>Sigma-Aldrich</td><td>A4034</td><td>Proximity-Labeling Quench Buffer, Lysis Buffer</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S8032</td><td>Proximity-Labeling Quench Buffer, Lysis Buffer, Flourescent Microscopy</td></tr><tr><td>Sodium chloride</td><td>Thermo Fisher Scientific</td><td>S271500</td><td>Cell Culture, Borate Buffer</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Thermo Fisher Scientific</td><td>BP1311220</td><td>Lysis Buffer, Dot blot assay buffer, Beads wash buffer</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>415413</td><td>Cell Culture, Borate Buffer</td></tr><tr><td>Sodium tetraborate</td><td>Sigma-Aldrich</td><td>221732</td><td>Cell Culture, Borate Buffer</td></tr><tr><td>SpeedVac concentrator</td><td></td><td></td><td>vacuum concentrator</td></tr><tr><td>Streptavidin Magnetic Sepharose Beads</td><td>Cytiva (formal GE)</td><td>28-9857-99</td><td>Enrich biotinylated proteins</td></tr><tr><td>Streptavidin, Alexa Fluor 680 Conjugate</td><td>Thermo Fisher Scientific</td><td>S32358</td><td>To conduct dot blot assay for bead titration</td></tr><tr><td>Thermomixer</td><td></td><td></td><td>temperature-controlled mixer</td></tr><tr><td>Trifluoacetic acid (TFA)</td><td>Millipore Sigma</td><td>302031</td><td>For Peptide desalting</td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td><td>Millipore Sigma</td><td>C4706</td><td>For Protein Digestion</td></tr><tr><td>Tris-HCl</td><td>Thermo Fisher Scientific</td><td>BP152500</td><td>Lysis Buffer, Dot blot assay buffer, Beads wash buffer</td></tr><tr><td>Triton-X</td><td>Thermo Fisher Scientific</td><td>BP151500</td><td>Beads wash buffer</td></tr><tr><td>TROLOX</td><td>Sigma-Aldrich</td><td>648471</td><td>Proximity-Labeling Quench Buffer, Lysis Buffer</td></tr><tr><td>Trypsin/Lys-C Mix, Mass Spec Grade</td><td>Promega</td><td>V5073</td><td>For Protein Digestion</td></tr><tr><td>TWEEN&amp;nbsp;20</td><td>Millipore Sigma</td><td>P1379</td><td>Dot blot assay buffer</td></tr><tr><td>Urea</td><td>Thermo Fisher Scientific</td><td>BP169500</td><td>Beads wash and On-Beads Digestion Buffer</td></tr><tr><td>Vitronectin</td><td>STEMCELL Technologies</td><td>7180</td><td>Cell Culture, Dish Coating</td></tr><tr><td>Y-27632 ROCK inhibitor</td><td>Selleck</td><td>S1049</td><td>Cell Culture</td></tr></tbody></table>

characterization of neuronal lysosome interactome with proximity labeling proteomics

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. 
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

This lysosome proximity labeling proteomics study was conducted in human iPSC-derived neurons to capture the dynamic lysosomal microenvironment in situ in live neurons. Cell morphologies of hiPSCs and hiPSC-derived neurons at different time points are illustrated in Figure 2A. Human iPSCs grow in colonies in E8 medium. Differentiation is initiated by plating iPSCs into doxycycline-containing neuron induction medium. Neurite extensions become more visible each day during the 3 day di...

Watch this Scientific Journal Video about Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics at JoVE.com

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to ...

characterization-neuronal-lysosome-interactome-with-proximity

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

2.4K Views.  The George Washington University. A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

Video: Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Biochemistry

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Biology

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

Immunology and Infection

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (&gt;40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at MCSs.

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium ...

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Summary

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Chapters in this video

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays