Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line
Summary
This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.
Abstract
Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.
Introduction
Aberrant protein homeostasis is caused by an imbalance in protein synthesis and degradation. Several diseases are related to alterations in protein homeostasis. The hallmark of some diseases is the presence of aggregates in different subcellular locations and brain areas. Protein homeostasis is not only important in disease but also plays a crucial role in normal organ and cellular function1. For example, protein synthesis is necessary for many forms of neuronal plasticity2,3, as determined by the use of chemical inhibitors that block protein synthesis4. However, it is neither clear in which cell-types the proteome is altered to support learning and memory, nor is it understood which specific proteins in each cell-type increase or decrease in their synthesis or degradation. Thus, a comprehensive study of protein homeostasis requires the capability to differentiate proteomes coming from specific cell-types. Indeed, the identification of cell-type-specific proteomes to study cellular processes occurring in a multicellular environment has been an important hurdle in proteomics. For this reason, we developed a technique using MetRS* expression combined with bio-orthogonal methods that has proven to be an effective way to identify and purify cell-type specific proteomes, filling this gap5,6,7.
The expression of a mutant MetRS* (MetRS L274G) allows for the loading of the non-canonical methionine analog ANL into the corresponding tRNA8,9 and its subsequent incorporation into proteins. When MetRS* expression is regulated by a cell-type-specific promoter, the non-canonical amino acid will be incorporated into the proteins in a cell-selective manner. Once ANL is incorporated in the proteins, it can be selectively functionalized by click-chemistry and subsequently either visualized by imaging (FUNCAT) or by Western Immunoblot (BONCAT). Alternatively, proteins can be selectively purified and identified by mass spectrometry (MS). Using this technology, we created a mouse line expressing the MetRS* protein under the control of the Cre recombinase. Considering the increasing number of available Cre-mouse lines, the MetRS* system can be used in any field to study any cell-type from any tissue for which there is an existing Cre-line. Protein labeling with ANL is possible in vitro or in vivo, and does not alter mouse behavior or protein integrity6. Labeling timespan can be adapted to the scientific question of each researcher, labeling newly synthesized proteins (shorter labeling times) or entire proteomes (longer labeling times). The use of this technique is limited by the number of cells of the type that the researcher is willing to study; hence protein isolation from cell-types with low numbers or low metabolic rates is not possible by this method. The goal of the presented method is to identify cell-type-specific proteins/proteomes labeled in vivo. In this protocol, we describe how to label cell-type-specific proteomes with ANL in live mice and purify the labeled proteins. After purification, proteins can be identified by routine mass spectrometry protocols5,10. The reduction of sample complexity achieved in this method by the selective purification of proteins from specific cellular populations allows the experimenter to detect subtle changes in proteomes, for example, in response to environmental changes. Purification of the labeled proteins can be achieved in ~10 days, not including the MS analysis or the labeling period. Here, we describe two methods for ANL administration to MetRS* expressing mice, namely (1) adding the amino acid in the drinking water, and (2) introducing ANL by intraperitoneal injections. Regardless of the method chosen for ANL administration, the isolation and purification steps are the same (from step 2 on).
Protocol
Representative Results
Discussion
The critical aspects of the protocol are; the inclusion of negative controls, having enough biological replicates, ANL administration route, amount, and duration, alkylation of the samples, alkyne concentration, and β-mercaptoethanol elimination when using DST-alkyne.
It is key to include negative control samples proceeding from ANL-labeled animals without Cre driver and therefore no MetRS* expression. These samples must be subjected to every step described in the protocol in parallel to …
Disclosures
The authors have nothing to disclose.
Acknowledgements
B.A-C is funded by the Spanish Ministry of Science and Innovation (Ramón y Cajal-RYC2018-024435-I), by the Autonomous Community of Madrid (Atracción de Talento-2019T1/BMD-14057), and MICINN (PID2020-113270RA-I00) grants. R. A-P is funded by Autonomous Community of Madrid (Atracción de Talento-2019T1/BMD-14057). E.M.S. is funded by the Max Planck Society, an Advanced Investigator award from the European Research Council (grant 743216), DFG CRC 1080: Molecular and Cellular Mechanisms of Neural Homeostasis, and DFG CRC 902: Molecular Principles of RNA-based Regulation. We thank D.C Dieterich and P. Landgraf for their technical advise and the synthesis of the DST-Alkyne. We thank E. Northrup, S. Zeissler, S. Gil Mast, and the animal facility of the MPI for Brain Research for their excellent support. We thank Sandra Goebbels for sharing the Nex-Cre mouse line. We thank Antonio G. Carroggio for his help with English editing. B.A-C. designed, conducted, and analyzed experiments. B.N-A, D.O.C, R.A-P, C. E., and S. t. D. conducted and analyzed experiments. B.A-C and E.M.S. designed experiments, and supervised the project, B.A-C wrote the paper. All authors edited the paper.
Materials
| 12% Acrylamide gels | GenScript | SurePAGE, Bis-Tris, 10 x 8, 12% | |
| β-Mercaptoethanol | Sigma | M6250 | Toxic; use a lab coat, gloves and a fume hood. |
| Ammonium bicarbonate | Sigma | 9830 | Toxic; use a lab coat, gloves and a fume hood |
| ANL | Synthesized as described previously for AHA (see references 5 and 11) | ||
| ANL-HCl | IrishBotech | HAA1625.0500 | |
| Benzonase | Sigma | E1014 | |
| Biotin alkyne | Thermo, | B10185 | |
| Chicken antibody anti-GFP | Aves | 1020 | |
| Complete EDTA-free protease inhibitor | Roche | 4693132001 | Toxic; use a lab coat and gloves. |
| Copper (I) bromide | Sigma | 254185 | 99.999% (wt/wt) |
| Disulfide tag (DST)-alkyne | Synthesized as reported in reference number 15, in which it is referred to as probe 20 | ||
| DMSO | Sigma | 276855 | |
| Filters | Merk | SCGP00525 | |
| Iodo acetamide (IAA) | Sigma | I1149 | |
| IR anti chicken 800 | LI-COR | IRDye 800CW | Donkey anti-Chicken Secondary Antibody |
| IR anti rabit 680 | LI-COR | IRDye 680RD | Goat anti-Rabbit IgG Secondary Antibody |
| Maltose | Sigma | M9171 | |
| Manual Mixer | BioSpec Products | 1083 | |
| NaCl | Sigma | S9888 | |
| N-ethylmaleimide | Sigma | 4259 | Toxic; use a lab coat, gloves and a fume hood. |
| NeutrAvidin beads | Pierce | 29200 | |
| Nitrocellulose membrane | Bio-rad | 1620112 | |
| PBS 1X | Thermo | J62036.K2 | |
| PBS 1X pH 7.8 | Preparation described in reference number 5 | ||
| PD SpinTrap G-25 columns | GE Healthcare | Buffer exchange | |
| Pierce BCA Protein Assay Kit | Thermo, | 23225 | Reagents in the Pierce BCA Protein Assay Kit are toxic to aquatic life. |
| Polyclonal rabbit anti-biotin antibody | Cell Signaling | 5597 | |
| PVDF membrane | Millipore | IPVH00010 | |
| SDS 10% | Sigma | 71736 | |
| SDS-PAGE Running buffer MOPS | GenScript | M00138 | |
| SYPRO Ruby stain | Sigma | S4942 | |
| Table automatic Vortexer | Eppendorf | Mixmate | |
| Triazole ligand | Sigma | 678937 | |
| Triton X-100 | Sigma | T9284 | |
| Water | Sigma | W4502 | Molecular biology grade |
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