Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein–protein interactions.
Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein–protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein–protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein–protein interactions.
Although proteins play constitutive roles in living organisms, they are continually synthesized, processed, and degraded in the intracellular environment. Furthermore, intracellular proteins frequently physically and biochemically interact, which affects the function of one or both1,2,3. For example, the direct binding of spliceosome-associated protein homolog CWC22 with eukaryotic translation initiation factor 4A3 (eIF4A3) is necessary for the assembly of the exon junction complex4. Consistent with this, an eIF4A3 mutant that lacks affinity for CWC22 fails to facilitate exon junction complex-driven mRNA splicing4. Thus, the study of protein interactions is crucial for the precise understanding of physiologic regulation as well as of cellular functions.
Recent advances in mass spectrometry (MS) have been applied to the comprehensive analysis of protein-protein interactions. For instance, the co-precipitation of endogenous proteins or exogenously introduced tagged proteins with their associated proteins, followed by MS analysis, enables the identification of novel protein interactions5. However, one major bottleneck of MS/MS analysis is poor recovery from tryptic digests of protein samples. For conducting proteomic analyses on cell lysates, in-gel and on-membrane digestion techniques are generally employed to prepare MS/MS samples. We have previously compared an in-gel digestion procedure with an on-membrane digestion technique6, and showed that the latter was associated with better sequence coverage. Polyvinylidene difluoride (PVDF) membrane may be suitable for this purpose because it is mechanically robust and resistant to high concentrations of organic solvents7,8, permitting the enzymatic digestion of immobilized proteins in the presence of 80% acetonitrile9. Furthermore, immobilization on a membrane can induce conformational changes in target proteins, leading to improvements in tryptic digestion efficiency10. Accordingly, in this article, we have described the use of immunoprecipitation-LC/MS/MS analysis of protein interactions using an on-membrane digestion technique. This simple method facilitates the convenient analysis of protein-protein interactions even in non-specialist laboratories.
1. Immunoprecipitation
NOTE: We used non-sodium dodecyl sulfate (SDS) lysis buffer and citrate elution, as described in the following sections. However, the use of an alternative in-house immunoprecipitation technique may be also applicable for preparing LC-MS/MS samples.
2. On-membrane digestion of proteins
NOTE: Using protein-free materials and equipment is necessary to avoid contamination with exogenous proteins. In addition, it is recommended that the operator wear a surgical mask and gloves to avoid contamination by human proteins.
3. LC–electrospray ionization (ESI)-MS/MS analysis
By means of the above-described procedure, immunoprecipitants were analyzed using LC-MS/MS (Figure 1). After the exclusion of exogenously derived proteins (proteins from other species and IgGs), 17 proteins were identified in calpain-6-associated immunoprecipitants (Table 1) and 15 proteins were identified in GFP-associated immunoprecipitants (Table 2). Of the calpain-6 and GFP-associated proteins, 11 were identified in both immunoprecipitants (Figure 2). Once these and calpain-6 itself were excluded, five candidate calpain-6-associated proteins remained: complement C1q subcomponent subunit C, keratin type II cytoskeletal 8, IgE-binding protein, ADP/ATP translocase 1, and ubiquitin.
Figure 1. Scheme for the on-membrane digestion technique
Cell lysates were precipitated using magnetic beads conjugated with specific antibodies. The eluant from the immunoprecipitation was blotted onto pieces of PVDF membrane. Subsequently, the membranes were treated with reagents for reductive alkylation, and then incubated with trypsin. The reaction solution was then analyzed using LC-MS/MS. Please click here to view a larger version of this figure.
Figure 2. Overview of the representative data
Seventeen proteins were identified in the calpain-6-associated immunoprecipitant and 15 proteins were detected in the GFP-associated immunoprecipitant. Of these, 11 proteins were identified in both immunoprecipitants. Please click here to view a larger version of this figure.
Name | Accession | Peptides(95%) | %Cov |
Actin, cytoplasmic 2 | sp|P63260|ACTG | 5 | 18.4 |
Actin, aortic smooth muscle | sp|P62737|ACTA | 5 | 18.57 |
Actin, cytoplasmic 1 | sp|P60710|ACTB | 5 | 18.41 |
Actin, alpha skeletal muscle | sp|P68134|ACTS | 5 | 13.53 |
Actin, alpha cardiac muscle 1 | sp|P68033|ACTC | 5 | 13.53 |
Actin, gamma-enteric smooth muscle | sp|P63268|ACTH | 5 | 13.56 |
Elongation factor 1-alpha 1 | sp|P10126|EF1A1 | 3 | 33.33 |
Elongation factor 1-alpha 2 | sp|P62631|EF1A2 | 3 | 16.2 |
Keratin, type II cytoskeletal 8 | sp|P11679|K2C8 | 3 | 22.65 |
Complement C1q subcomponent subunit C | sp|Q02105|C1QC | 2 | 8.94 |
IgE-binding protein | sp|P03975|IGEB | 2 | 21.72 |
Calpain-6 | sp|O35646|CAN6 | 1 | 15.91 |
ADP/ATP translocase 2 | sp|P51881|ADT2 | 1 | 27.52 |
ADP/ATP translocase 1 | sp|P48962|ADT1 | 1 | 19.13 |
Ubiquitin | sp|P62991|UBIQ | 1 | 40.79 |
Runt-related transcription factor 3 | sp|Q64131|RUNX3 | 1 | 15.89 |
Isoform 2 of Runt-related transcription factor 3 | sp|Q64131-2|RUNX3 | 1 | 13 |
“Peptides (95%)” indicates the number of peptides identified with a fidelity score > 95% in the MS/MS data. “%Cov” refers to the percentage of the amino acid residues identified in all the peptides (with > 95% fidelity) relative to the total number of amino acid residues constituting the corresponding protein. To improve clarity, exogenous proteins (proteins from other species and IgGs), ribosomal proteins, and histones are not shown. |
Table 1. Calpain-6-associated proteins
Name | Accession | Peptides(95%) | %Cov |
Elongation factor 1-alpha 1 | sp|P10126|EF1A1 | 3 | 24.24 |
Elongation factor 1-alpha 2 | sp|P62631|EF1A2 | 3 | 24.41 |
Actin, alpha skeletal muscle | sp|P68134|ACTS | 3 | 13.53 |
Actin, alpha cardiac muscle 1 | sp|P68033|ACTC | 3 | 13.53 |
Actin, gamma-enteric smooth muscle | sp|P63268|ACTH | 3 | 13.56 |
Actin, cytoplasmic 2 | sp|P63260|ACTG | 3 | 13.6 |
Actin, aortic smooth muscle | sp|P62737|ACTA | 3 | 13.53 |
Actin, cytoplasmic 1 | sp|P60710|ACTB | 3 | 13.6 |
ADP/ATP translocase 2 | sp|P51881|ADT2 | 2 | 25.5 |
rRNA 2'-O-methyltransferase fibrillarin | sp|P35550|FBRL | 2 | 14.37 |
Prohibitin | sp|P67778|PHB | 1 | 9.19 |
Heterogeneous nuclear ribonucleoprotein U | sp|Q8VEK3|HNRPU | 1 | 10.63 |
Runt-related transcription factor 3 | sp|Q64131|RUNX3 | 1 | 39.12 |
Isoform 2 of Runt-related transcription factor 3 | sp|Q64131-2|RUNX3 | 1 | 26 |
Elongation factor 1-gamma | sp|Q9D8N0|EF1G | 1 | 12.36 |
“Peptides (95%)” indicates the number of peptides identified with a fidelity score > 95% in the MS/MS data. “%Cov” refers to the percentage of the amino acid residues identified in all the peptides (with > 95% fidelity) relative to the total number of amino acid residues constituting the corresponding protein. To improve clarity, exogenous proteins (proteins from other species and IgGs), ribosomal proteins, and histones are not shown. |
Table 2. GFP-associated proteins
We have previously described an analysis of the oxidative modifications of apolipoprotein B-100 in oxidized low-density lipoprotein using LC-MS/MS preceded by an on-membrane digestion technique6. In the present study, we combined this technique with immunoprecipitation and have identified several calpain-6-associated proteins. This novel technique represents a convenient method of screening for candidate associated proteins. Calpain-6 is a non-proteolytic member of the calpain proteolytic family11 that has reportedly modify cellular functions through its protein-protein interactions12,13. Using an on-membrane digestion technique, we have previously identified other calpain-6-associated proteins employing a different MS set-up12. Therefore, we recommend testing several MS conditions for maximizing the number of candidates identified. For the same reason, the evaluation of the immunoprecipitation conditions, such as the types of epitope tag, detergent, and elution solution used, is important for obtaining optimal outputs.
In the present study, we identified 11 proteins in both calpain-6- and GFP-associated immunoprecipitants. The majority of the overlapping proteins were actin isotypes, and contamination with actin cytoskeletal proteins is common in the analyses of cellular protein-protein interactions because the expression levels of these proteins are very high. These candidates should therefore be omitted from further analysis. In addition, elongation factors were detected in both immunoprecipitants. They regulate the speed and fidelity of protein synthesis and affect protein-folding14, and, therefore, the co-immunoprecipitation of elongation factors is not surprising. These proteins should also be omitted from further evaluation.
Immunoprecipitants can be subjected to reductive alkylation and enzymatic digestion directly in the elution solution15, and this in-solution digestion method may also be applied for the preparation of samples for MS/MS. However, we consider the use of on-membrane digestion to have considerable advantages over in-solution digestion. PVDF membrane serves as a scaffold for the subsequent reductive alkylation and enzymatic digestion, meaning that the solvents required for these processes can be replaced easily. Consequently, it is possible to use a variety of elution solutions for immunoprecipitation. Conversely, for in-solution digestion, it may be challenging to use SDS-based or low pH elution solutions because protease activity may be limited under such conditions. Furthermore, immobilization of the target proteins can make the subsequent washing procedure easier. Hence, on-membrane digestion is highly suitable for the preparation of immunoprecipitants for MS/MS analysis. A limited number of proteases may be appropriate for this protocol. Thus far, only Lysyl-C, other than trypsin, is reportedly active in the presence of up to 80% acetonitrile6,9,10,15.
Our on-membrane digestion technique is suitable for the identification of proteins in a small quantity of immunoprecipitant with a highly sensitive detection limit. However, it should be remembered that the detection efficiency for a target protein depends on the amount present. Proteins that are present in larger quantities are preferentially detected and they may prevent other proteins present in smaller amounts being detected by MS. Nevertheless, under normal circumstances numerous proteins are detected using LC-MS/MS analysis, and other assays must be used for clarify which of the candidate proteins are of appropriate biologic significance.
In this study, we have evaluated an on-membrane digestion technique for the analysis of immunoprecipitants. Such a convenient and comprehensive method for the analysis of protein-protein interactions should be widely applicable to improve the throughput of future proteomic analyses.
The authors have nothing to disclose.
This study was supported in part by Japan Society for the Promotion of Science KAKENHI Grant Number 17K09869 (to AM), Japan Society for the Promotion of Science KAKENHI Grant Number 15K09418 (to TM), a research grant from Kanehara Ichiro Medical Science Foundation and a research grant from Suzuken Memorial Foundation (all to TM).
Acetonitrile | Wako | 014-00386 | |
Citric acid | Wako | 030-05525 | |
DiNA | KYA Tech Co. | nanoflow high-performance liquid chromatography | |
DiNa AI | KYA Tech Co. | nanoflow high-performance liquid chromatography equipped with autosampler | |
DTT | Nacalai tesque | 14112-94 | |
Dynabeads protein G | Thermo Fisher Scientific | 10003D | |
Formic acid | Wako | 066-00461 | |
HiQ Sil C18W-3 | KYA Tech Co. | E03-100-100 | 0.10mmID * 100mmL |
Iodoacetamide | Wako | 095-02151 | |
Lipofectamine 3000 | Thermo Fisher Scientific | L3000008 | |
Living Colors A.v. Monoclonal Antibody (JL-8) | Clontech | 632380 | |
NaCl | Wako | 191-01665 | |
NH4HCO3 | Wako | 018-21742 | |
Nonidet P-40 | Sigma | N6507 | poly(oxyethelene) octylphenyl ether (n=9) |
peptide standard | KYA Tech Co. | tBSA-04 | tryptic digests of bovine serum albumin |
PP vial | KYA Tech Co. | 03100S | plastic sample tube |
Protease inhibitor cooctail | Sigma | P8465 | |
ProteinPilot software | Sciex | 5034057 | software for protein identification |
Sequencing Grade Modified Trypsin | Promega | V5111 | trypsin |
Sodium orthovanadate | Sigma | S6508 | |
Sodium phosphate dibasic dihydrate | Sigma | 71643 | |
TFA | Wako | 206-10731 | |
trap column | KYA Tech Co. | A03-05-001 | 0.5mmID * 1mmL |
TripleTOF 5600 system | Sciex | 4466015 | Hybrid quadrupole time-of-flight tandem mass spectrometer |
Tris | Wako | 207-06275 | |
Tween-20 | Wako | 160-21211 |