Examine signaling pathways with targeted proteomics

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The AKT/mTOR pathway plays a central role in tumor progression and cancer-drug resistance, and therefore the quantitative measurement of this pathway’s protein expression and posttranslational modifications (PTMs) is vital to cancer research [1]. A major limitation when measuring AKT/mTOR pathway protein levels is the lack of rigorously validated methods and reagents, as well as a reliance on semiquantitative results from western blot analyses. Because many biologically relevant proteins are present in vanishingly small quantities, immunoprecipitation (IP) is commonly used as a tool for enriching protein [2,3]. Mass spectrometry (MS) is increasingly becoming the detection method of choice for determining protein abundance and identifying PTMs.

The IP-MS workflow, which combines IP steps with subsequent analysis by MS, can be used to enrich signaling proteins, benchmark antibody performance, and reveal protein– protein interactions [4]; see “Comprehensive strategy for antibody validation” in BioProbes 75 Journal of Cell Biology Applications. Multiplex IP coupled with targeted MS (mIP-tMS) further enhances this workflow by simultaneously quantifying multiple proteins and their phosphorylation states in a specific signaling pathway. Here we demonstrate the mIP-tMS methodology by analyzing a specific set of protein targets in the AKT/mTOR pathway.

Singleplex IP-MS assay development for AKT/mTOR pathway proteins

To begin our analysis of the AKT/mTOR pathway, we selected 11 pathway proteins—10 of which have at least one phosphorylation site—for antibody validation by IP-MS (Table 1). These targets were chosen to align with commercially available reagents for western blot analysis (WB), enzyme-linked immunosorbent assays (ELISA), and Luminex® bead-based multiplex immunoassays (Luminex assays). Figure 1 shows the general workflow for mIP-tMS assay development.

The first step was to validate antibodies by IP-MS and identify quantotypic peptides for each AKT/mTOR pathway protein. Antibody candidates were screened by IP-MS to determine their effectiveness—both their ability to immunoprecipitate AKT pathway proteins and their usefulness when combined with MS. The IP-enriched samples were then analyzed by LC-MS to qualitatively identify targets of interest, interacting proteins, PTMs, and quantotypic peptides. We performed the LC-MS analysis using a Thermo Scientific Dionex UltiMate 3000 RSLCnano System and Thermo Scientific Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer, and the data were analyzed with Thermo Scientific Proteome Discoverer 1.4 software to assess percent sequence coverage, unique peptides, area, and PTMs for each pathway protein.

We identified the unique tryptic peptides with the best analytical characteristics for each protein target, and selected these quantotypic peptide sequences for the synthesis of stable isotope–labeled AQUA peptides (using HeavyPeptide AQUA Ultimate custom services) that serve as internal standards. Heavy AQUA peptides for each protein target were further evaluated for linearity, reproducibility, accuracy, dynamic range, quantitation limits, and recovery.

Finally, absolute quantitation of each protein target was achieved by utilizing a standard curve with serial dilutions of each heavy AQUA peptide across three orders of magnitude, in conjunction with targeted MS (tMS) using the parallel reaction monitoring (PRM) method implemented on the mass spectrometer. The tMS data were analyzed with Skyline software (MacCoss Lab Software, University of Washington) to determine the limit of quantitation (LOQ) from the calibration curve and the target analyte concentration from unknown samples.

Table 1. Total and phosphorylated AKT/mTOR pathway protein targets selected for analysis.

Protein target Phosphorylation site
AKT1/AKT2 pSer473/pSer474
mTOR pSer2448
IGF1R pTyr1135/1136
IR NA
PRAS40 pThr246
p70S6K pThr389
TSC2 pSer939
PTEN pSer380
GSK3α pSer21
GSK3β pSer9
IRS1 pSer312

Figure 1. mIP-tMS assay workflow. Multiple antibodies for the 11 total and 10 phosphorylated AKT/mTOR pathway protein targets were validated using the Thermo Scientific Pierce MS-Compatible Magnetic IP Kit (protein A/G) and analyzed by LC-MS. The antibody with greatest capture efficiency for each target was then selected for biotinylation using the Thermo Scientific Pierce Antibody Biotinylation Kit for IP. Lastly, the 11 total and 10 phosphorylated protein targets were simultaneously enriched by mIP using validated biotinylated antibodies and the Thermo Scientific Pierce MS-Compatible Magnetic IP Kit (streptavidin). mIP eluate samples were processed by an in-solution digestion method to generate a proteotypic peptides mix. Internal standard heavy peptide mix for 11 AKT/mTOR pathway proteins was spiked into digested peptides, and tMS assay was performed in parallel reaction monitoring (PRM) mode for quantitation of 11 pathway proteins in a single MS run.

Enrichment of AKT/mTOR pathway protein targets

Table 2 shows the enrichment of low-abundance AKT/mTOR pathway protein targets by the singleplex IP-MS method. IGF stimulation has been shown to activate AKT/mTOR pathway signaling through phosphorylation cascades [5]. AKT/mTOR pathway protein targets were immunoprecipitated from unstimulated and IGF-stimulated A549 lysates for MS analysis using the IP-MS methodology described above. When compared with neat lysates (combined lysates from the unstimulated and IGF-stimulated cells that were not immunoprecipitated), the IP-enriched samples and LC-MS analysis allowed us to identify a significantly larger number of unique peptides (Table 2).

The effect of IGF stimulation on AKT/mTOR pathway proteins was assessed by comparing the abundance of signaling proteins, interacting partners, and PTMs for each protein target. Protein isoforms and interacting partners were identified for total AKT, IGF1R, and mTOR targets. Relevant phosphorylation sites were detected for phosphorylated AKT1, AKT2, mTOR, IGF1R, and PRAS40 targets. Relative abundance of each pathway protein was determined using the total area of all unique peptides identified for each target. A larger number of unique peptides for phosphorylated AKT and IGF1R were observed for IGF-stimulated A549 lysates compared with unstimulated samples. These results demonstrate that beyond simply identifying protein targets from whole lysates, the singleplex IP-MS method can also be applied to interrogate AKT/mTOR pathway signaling events in the context of IGF stimulation.

Table 2. Enrichment of low-abundance AKT/mTOR pathway proteins by IP-MS.

IP antibody Targets identified Neat no. of unique peptides IP-enriched no. of unique peptides Relevant phospho-peptide ID
–IGF +IGF
Phospho-AKT AKT1 3 20 +IGF: Ser473
AKT2 14 +IGF: Ser474
AKT3 13 NA
AKT1 AKT1 16 12 NA
AKT2 9 11 NA
AKT3 5 3 NA
Phospho-mTOR mTOR 2 75 82 Thr2446, Ser2448
RICTOR 2 NA
SIN1 2 3 NA
Gbl 4 4 NA
IGF1R IGF1R 4 13 13 NA
IR 10 6 NA
Phospho-IGF1R IGF1R 4 5 +IGF: Tyr1135/1136
PRAS40 PRAS40 8 8 Thr246
Phospho-PRAS40 PRAS40 8 6 Thr246

Validation of mIP-tMS assays

The singleplex IP-MS analysis described above highlighted antibodies that successfully enrich their intended targets, interacting proteins, and PTMs; however, it required that we perform separate IPs for each of the 10 phosphorylated (and 11 total) protein targets from the AKT/mTOR pathway. Alternatively, mIP-tMS analysis should allow simultaneous enrichment and quantitation of all targets from a single IP. Focusing on the AKT/mTOR proteins that we identified earlier, we validated the use of 10-plex phospho and 11-plex total mIP-tMS assays with unstimulated and IGF-stimulated MCF7 lysates. mIP was carried out using biotinylated antibodies and the Thermo Scientific Pierce MS-Compatible Magnetic IP Kit (streptavidin). For the tMS analysis, standard curves were generated using 18 spiked-in AQUA heavy peptides for 12 targets (including both AKT1 and AKT2) and then employed for absolute quantitation of AKT/mTOR proteins. These mIP-tMS assays identified 11 proteins in the multiplex phospho assay and 12 proteins for the multiplex total assay (Table 3). We found that in addition to the IRS1 protein, PI3K subunits were identified in IGF-stimulated MCF7 cell lysates. Upregulation of phosphorylated AKT1, AKT2, and IGF1R was observed upon IGF stimulation.

Table 3. Validation of phospho and total mIP-tMS assays.

Target 10-plex phospho assay 11-plex total assay
Number of unique peptides
–IGF +IGF –IGF +IGF
AKT1 9 25 30
AKT2 4 24 26
mTOR 48 56 25 28
IGF1R 1 3 32 35
IR NA NA 29 26
PRAS40 5 7 9 10
p70S6K 9 14 11 12
TSC2 5 10 42 45
PTEN 1 5 9
GSK3α 7 6 19 21
GSK3β 13 10 23 23
IRS1 4 11 45 54
PIK3R1 22
PIK3CA 2
PIK3CB 6
PIK3R2 22

Benchmarking mIP-tMS assays

Several singleplex or multiplex immunoassay-based techniques exist for quantifying proteins of interest from biological samples. We sought to compare the mIP-tMS assay with established immunoassays in order to benchmark its analytical performance. We analyzed 11 phosphorylated and 12 total AKT/mTOR pathway proteins in unstimulated and IGF-stimulated HCT116, A549, and MCF7 cells using mIP-tMS, WB, ELISA, and Luminex assays (Figure 2). mIP-tMS assays allowed absolute quantitation for all 12 total and 11 phosphorylated targets in low- to subnanogram concentrations across all cell lines. The comparative summary of the four techniques showed target-dependent correlation, and the WB method disagreed most often with the other three methods (Figure 2). Variability across techniques for some targets can be the result of antibody specificity. Phosphorylated targets showed lower correlation compared to total protein abundances from the AKT/mTOR pathway.

A
B

Figure 2. Technology benchmarking of the AKT/mTOR pathway. Comparison of mIP-tMS assays with current immunoassay techniques to quantitate AKT-mTOR pathway protein targets from unstimulated and IGF-stimulated A549, HCT116, and MCF7 lysates were performed. Western blot, ELISA, and Luminex assays were performed according to manufacturer’s instructions. mIP-tMS assays for 11 total and 10 phosphorylated targets were performed in PRM mode using the Thermo Scientific Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer. Good correlation was observed for (A) total AKT and (B) phospho IGF1R for mIP-tMS assays, ELISA, and Luminex assays but not western blot. RQ = relative quantitation; AQ = absolute quantitation.

Conclusions

Using the mIP-tMS method developed for the AKT/mTOR pathway, we verified antibody selectivity and assessed interactions and off-targets for AKT/mTOR pathway proteins. Validated antibodies and optimized mIP-tMS workflows combine the benefits of target enrichment, selectivity, and flexibility to better interrogate complex biological interactions. The multiplexing capabilities of mIP-tMS assays provide an effective strategy for increasing sample throughput without sacrificing detection limits.

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