Immunoprecipitation (IP) stands as a classical experimental technique, commonly paired with SDS-PAGE and Western Blotting (WB) for protein detection. Consequently, when researchers design IP-MS experiments for screening interacting proteins, they often draw upon their prior experiences with IP-WB experiments and the conventional sample preparation methods for in-gel enzymatic digestion from traditional proteomic studies. Unfortunately, in doing so, they may overlook the need to adapt and optimize experimental methods and strategies in light of advancements in mass spectrometry instrument performance and data analysis methodologies. This oversight can lead to less-than-ideal detection results for protein interactions.
This article has compiled recommendations for the selection of IP experimental samples and various materials tailored specifically for protein interaction studies based on mass spectrometric detection. Our aim is to provide guidance and assistance to researchers in related fields, urging consideration and adaptation to current advancements in mass spectrometry instruments and data analysis methods for optimal experimental outcomes.
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Cell Quantity in IP-MS Experiments
The IP-MS experiment typically commences with the lysis of cellular or tissue samples. In comparison to Western blot (WB) experiments, which employ cascading amplification reactions, the sensitivity of mass spectrometry detection is generally slightly lower. Furthermore, unlike the specific detection of target proteins in WB, mass spectrometry theoretically detects all protein components in the sample. Consequently, IP experiments used for mass spectrometry detection typically require a higher initial cell quantity compared to traditional IP-WB.
The initial cell quantity for IP-MS experiments is primarily adjusted based on factors such as cell type, abundance of the target protein, and the efficacy of the affinity antibody. It is generally recommended that the starting cell quantity per sample should not be less than 2e7. If the cell line expresses the target protein at a high level, such as overexpressed tagged proteins or endogenous proteins with high expression within the cells (e.g., Actin, RPS, etc.), the cell quantity can be appropriately reduced but is recommended not to be less than 1e7. Within a certain range, increasing the cell quantity enhances the quality of the obtained mass spectrometry results.
For experiments utilizing human cells or tissues, the abundance of the target protein can be referenced from the Proteomics DB (https://www.proteomicsdb.org/) and Human Protein Atlas (https://www.proteinatlas.org/) databases. The former is derived from publicly available mass spectrometry detection data, while the latter primarily relies on protein expression information from RNA sequencing and antibody-related experimental data.
Table 1: Cellular Quantities in Published IP-MS Studies
| Publication Source | Cell Type | Cell Quantity |
|---|---|---|
| Cell Rep (2012) | Yeast | 5e11 |
| Cell Rep (2013) | Mouse | 3e9 |
| Nat.Struct.Mol.Biol. (2013) | Drosophila S2 Cells | 5e9 |
| J.Vis.Exp. (2014) | HeLa | 1-2e8 |
| Nat.Commun (2015) | Mouse ESCs | 3e8 |
| PNAS (2015) | Mouse ESCs | 6-8e8 |
| Cell (2015) | HeLa | 5e7 |
| Cell Rep (2017) | Mouse CD4+ T cells | 1e8 |
| Proteomics (2018) | THP-1 cells | 1e8 |
| Nature (2018) | hESC | 2e7 |
Antibody Selection
Antibodies stand as the pivotal determinant of the success of immunoprecipitation (IP) experiments. Currently, commonly used tag antibodies generally exhibit robust specificity and potency, satisfying the experimental requirements of IP-MS and yielding commendable detection outcomes. However, the effectiveness of commercially available antibodies targeting various endogenous proteins is often challenging to guarantee.
Studies have systematically assessed the performance of thousands of commercial antibodies, revealing that less than a quarter achieve satisfactory results in Western blot (WB) experiments, with nearly half providing inconclusive outcomes. Generally, the demands on antibodies in IP experiments surpass those in WB. To mitigate false-positive interaction identifications stemming from antibody nonspecificity, antibodies used in IP-MS undergo higher scrutiny compared to traditional IP-WB experiments.
Therefore, antibodies designated for IP-MS experiments are advised to undergo a meticulous four-step selection and validation process:
Preferentially select antibodies explicitly marked for use in IP experiments and those employed by other researchers.
Investigate results provided by antibody manufacturers and prior users, eliminating antibodies with significant discrepancies in band size compared to the theoretical size of the target protein, as well as those exhibiting nonspecific bands and excessive background.
Conduct IP-WB pre-experiments upon antibody procurement, assessing antibody performance. Prioritize antibodies with band sizes consistent with the target protein and devoid of other nonspecific bands.
Employ mass spectrometry detection to identify proteins in a small subset of IP-MS samples, selecting antibodies with detection signals for the target protein ranking prominently. In certain scenarios, antibodies provided by manufacturers may specifically recognize other proteins with molecular weights close to, but different from, their labeled targets—a circumstance challenging to validate through IP-WB experiments.
Affinity Resins
Affinity resins, often referred to as "beads" in immunoprecipitation (IP) experiments, typically consist of two components: the matrix material and the IgG-binding protein. Currently, there are two commonly used matrix materials: agarose/sepharose and magnetic beads. In comparison, magnetic beads exhibit higher affinity, simplified handling, and improved washing efficacy but come at a slightly higher cost than agarose beads. In the context of IP-MS experiments, the use of magnetic beads is strongly recommended.
The IgG-binding proteins within beads are generally categorized as Protein A, Protein G, and Protein A/G. For the most commonly employed mouse and rabbit antibodies in IP experiments, both Protein A and Protein G can effectively bind. However, there is a slight difference in their binding capabilities for antibodies from other species. Protein A/G is a fusion protein containing both Protein A and Protein G antibody-binding domains, effectively binding to the majority of antibodies used in IP experiments.
In cases where affinity purification is directed towards tagged proteins, most commercially available tag-affinity beads currently on the market can achieve excellent affinity purification results without the need for additional purchase of tagged antibodies for incubation.
Lysis Buffer, Washing Buffer, and Elution Buffer
The lysis buffer typically comprises three main components: a detergent, protease inhibitors, and a buffering system.
In the lysis buffer for immunoprecipitation (IP), the detergent component is commonly 0.5%-2% NP40 or TritonX-100, and a combination of both may also be used. To enhance lysis efficiency, some lysis buffers may contain a 0.01% concentration of SDS or SDC. It is noteworthy that the use of high concentrations of SDS (>0.1%), commonly found in other lysis buffers, can severely disrupt protein-protein interactions and is thus generally unsuitable for IP experiments.
Since IP experiments involve incubation, protein degradation is a concern, necessitating the addition of protease inhibitors to the lysis buffer. Additionally, phosphatase inhibitors or broad-spectrum nucleases may be added based on the target protein type and specific research objectives.
The common buffering system in IP buffer is 150 mM NaCl/50 mM Tris-HCl, pH 7.5. While slight variations in salt concentration and pH may exist among different research groups, it is crucial to note that high salt concentrations (>300 mM NaCl) can significantly disrupt weaker protein interactions, leading to a drastic reduction in protein identification results during mass spectrometry detection.
In conventional IP-WB experiments, the lysis buffer is typically used directly as the washing buffer, and a loading buffer containing high concentrations of SDS, bromophenol blue, and β-mercaptoethanol is employed as the elution buffer. However, in IP-MS experiments, to better align with subsequent sample processing for mass spectrometry detection, it is essential for the post-washing sample to be free from detergent components. Therefore, it is recommended to use a detergent-free buffering system or PBS as the washing buffer. Alternatively, after conventional washing, PBS or a buffering system can be used for a final wash to replace the detergent-containing components. To retain interacting proteins in the sample as much as possible and minimize protein loss during sample preparation, it is advisable to directly precipitate the beads for subsequent sample preparation for mass spectrometry detection after washing, without eluting the proteins.
Summary
The outcome of IP-MS experiments is significantly contingent upon the effectiveness of the immunoprecipitation (IP) step. However, in contrast to more conventional IP-WB experiments conducted in laboratories, the IP component of IP-MS experiments entails specific requirements, primarily:
(1) Larger cell quantities are recommended, with a suggested minimum of 2e7 cells. (2) Higher demands for antibody specificity and affinity are crucial. Preferably, use tagged antibodies or those validated for IP efficacy. (3) Post-IP, it is essential to wash beads to remove detergents like NP40. PBS or similar solutions are suitable as washing buffers.
References
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