Automating PTM Profiling for Mass Spec: Scalable Proteomics Workflows Using PTMScan^® Reagents

Studying post-translational modifications (PTMs) is critical for advancing our understanding of cell signaling, disease biology, and therapeutic mechanisms. With the ability to measure hundreds to tens of thousands of PTM sites per sample, PTMScan technology from CST is a robust and versatile method that has been used to identify novel biomarkers in many contexts, including drug discovery and characterization.^1-4 As researchers look to leverage larger sample cohorts in drug development pipelines, incorporating programmable automation into PTMScan workflows can increase throughput and reproducibility, enabling the type of quantitative data that leads to actionable—and reliable—biological insights.

This blog explores how PTMScan reagents can be used in automation-based workflows and the benefits for quantitative, high-throughput proteomics studies.

Beyond ELISA: High-Throughput PTM Detection with Automated LC-MS

Automation platforms are commonly used for ELISA-like assays or high-content imaging applications, where they typically measure a small number of variables at once, often just one PTM site per sample.

In contrast, PTMScan workflows combine the PTM enrichment capabilities of PTM- or PTM-motif-specific immunoaffinity beads with the quantitative measurement afforded by liquid chromatography-mass spectrometry (LC-MS) to enable the analysis of hundreds to tens of thousands of PTM sites per sample, far exceeding what is typically possible with high-content imaging assays

Figure 1. Scope of data generated using PTMScan HS Phospho-Tyrosine (P-Tyr-1000) Kit #38572, showing increasing depth of analysis. A) Western blot analysis of global phosphotyrosine (pY) levels in A431 cells, untreated (-) and treated (+) with EGF ligand. B) Tally of unique pY sites identified by PTMScan HS PhosphoTyrosine from control and EGF-treated samples. C) Overlap of unique pY sites identified in control and EGF-treated samples. D) Quantification of pY site abundances in control and EGF-treated samples. 

However, one of the primary bottlenecks in quantitative proteomics workflows has historically been the manual nature of LC-MS analysis. While advances in the speed, resolution, and sensitivity of MS technologies, such as with data-independent acquisition (DIA) proteomics, have reduced the dependence on lengthy acquisitions, the enrichment step still remains a challenge. When performed manually, bead-based PTM enrichment is time-consuming and can introduce variability across samples. It is this stage of the workflow where automation offers the most benefit.

We tested PTMScan reagents on some of the most commonly used commercial platforms (Table 1) for high-throughput affinity enrichment processing to ensure compatibility and measure the effect on experimental reproducibility.

In our testing, automation far outperformed manual workflows—for example, peptide identification was 30-135% higher on the AssayMAP Bravo System compared to manual preparation. Additionally, for the magnetic bead-based PTMScan HS reagents, enrichment when automated on the KingFisher Apex performed as well as enrichment when performed manually, but with greater handling ease from the user and scalability to larger sample sizes.

Read on for more details about our findings on different platform types, and to review example references from the literature where the authors leveraged PTMScan reagents in automated workflows.

Bead-Handler Platforms

Arguably the most user-accessible platform is the bead-handler type. Because the beads used in PTMScan HS reagents are relatively large, they can be quickly captured and moved by the magnetic probe, making them well-suited for bead-handler automation robots.

To test performance, we compared a manual vs an automated ubiquitin enrichment experiment using our PTMScan HS Ubiquitin/SUMO Remnant Motif (K-ε-GG) Kit #59322 and the ThermoFisher KingFisher Apex system. We found similar recovery of PTM peptides between automated and manual experiments. 

Using PTMScan HS Kits on the KingFisher Apex

• Methodology & Experiment Set-Up: In a typical setup, magnetic beads, peptide samples, and wash/elution buffers are distributed across plates, with each well assigned to a sample-enrichment combination. Using an in-house-developed KingFisher Apex protocol, we performed a ubiquitin enrichment experiment with our PTMScan HS Ubiquitin/SUMO Remnant Motif (K-ε-GG) #59322 kit on two different dates. On each date, we performed a manual or KingFisher-based enrichment across three biological replicates (i.e., three parallel enrichments) from the same input peptide type (i.e., mouse liver peptides).  
• Results: We observed similar recovery of PTM peptides between automated and manual experiments. 

图 2. The reproducibility of data generated from a PTMScan HS Ubiquitin experiment, where the enrichment was performed manually or with a KingFisher Apex robot. A) Number of unique PTM peptides identified. B) Overlap of ~1000 randomly selected PTM peptides between Manual and KingFisher workflows. C) Label-free quantification of MS1 peak area (natural log transformed) of PTM peptides shared between Manual and KingFisher workflows.

You can also explore the following papers, where researchers used PTMScan reagents in high-throughput workflows:

	Automating UbiFast for High-throughput and Multiplexed Ubiquitin Enrichment Rivera et al. (Mol Cell Proteomics 2021) | DOI: 10.1016/j.mcpro.2021.100154 The authors present a method of robotic automation using a magnetic bead-conjugated K-ε-GG antibody  (PTMScan HS Ubiquitin/SUMO Remnant Motif (K-ε-GG) Kit #59322) on the KingFisher Flex
	Automated Immunoprecipitation Workflow for Comprehensive Acetylome Analysis Gritsenko et al. (Methods Mol Bio. 2024) | DOI: 10.1007/978-1-0716-3922-1_12 The authors describe a fast, automated enrichment protocol that enables reproducible and comprehensive acetylome analysis using a magnetic bead-based immunoprecipitation reagent (PTMScan HS Acetyl-Lysine Motif (Ac-K) Kit #46784) on the KingFisher Flex.
	Androgen drives melanoma invasiveness and metastatic spread by inducing tumorigenic fucosylation Liu et al. (Nat Commun 2014) | DOI: 10.1038/s41467-024-45324-w To interrogate the role of the male sex hormone androgen and its receptor (AR) in melanoma, the authors used PTMScan Phospho-Enrichment IMAC Fe-NTA Magnetic Beads #20432 on the KingFisher Flex.
	Automated PTMScan immunoaffinity enrichment for the capture of KGG modified peptides from complex mixtures Phu et al. (ASMS 2019) | DOI: 10.13140/RG.2.2.11593.88164 This poster outlines a protocol for performing PTMScan immunoaffinity enrichment on the Phynexus MEA robot, a robust automation platform that enables the concurrent processing of up to twelve samples.

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Hybrid Platforms

PTMScan kits are also compatible with hybrid automation systems, such as the Agilent AssayMAP Bravo system. Unlike bead-handling systems, this platform uses specialized tips—typically pre-loaded with Protein A—onto which antibodies from the PTMScan kit can be immobilized. Rather than incubating the PTMScan bead-based slurry with a peptide solution or wash buffers, in this type of hybrid platform, the liquid handler pulls liquid through the tips containing the antibodies.  

This approach offers greater experimental flexibility and broader compatibility across a range of PTMScan-validated antibodies, including those that are not conjugated to magnetic beads. While the magnetic bead-based PTMScan HS products are not conducive for hybrid platforms, PTMScan-validated non-bead-conjugated antibodies are amenable to this highly reproducible and convenient platform.

Alissa Nelson Principal Scientist, Proteomics

作者信息： This type of platform can be susceptible to clogged tips. Users can minimize this risk by sonicating peptide samples in a water bath and then centrifuging at 10,000xg for 5 minutes to remove any insoluble microparticulates prior to PTM enrichment.

To evaluate the performance of PTMScan reagents on the AssayMAP Bravo platform, we compared manual agarose bead-based enrichments to on-tip enrichment for three antibody targets. We found that the bidirectional aspirate program on AssayMAP Bravo delivered the best results, including significantly higher peptide identification compared to manual preparation. 

Using PTMScan Reagents on the Agilent AssayMAP Bravo System

Methodology & Experimental Set-Up: We compared manual agarose bead-based enrichments to on-tip enrichment for three well-studied PTMs: Acetyl-lysine using PTMScan Acetyl-Lysine Motif [Ac-K] Kit #13416; ubiquitin using PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit #5562, and phospho-tyrosine using PTMScan Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) Kit #8803.
Immunoprecipitations (IPs) were performed on two separate days, and LC-MS samples were acquired on a Thermo Scientific Q-Exactive system. The Agilent AssayMAP Bravo software offers two general workflows for antibody purification: 

• A bidirectional aspirate program, where peptides are drawn up through the bottom of the tip prior to dispensing them into a flow-through collection plate.
• A unidirectional aspirate program (dispense-only) draws the samples with bare probe tips and then dispenses the peptides through the tips from the top.

Results: We found that the bidirectional aspirate program significantly outperformed both manual preparation and the unidirectional dispense-only program—PTM peptide identifications were 30-135% higher with the bidirectional aspirate program compared to manual preparation, and the dispense-only program performed more poorly than manual.

图 3. An example illustrating the reproducibility of data generated from various PTMScan experiments, where the enrichments were performed manually or with an AssayMAP Bravo robot. Note the improved recovery of PTM peptides when the workflow is performed via the automation platform.

Need custom PTMScan products? Click here to inquire about lot reservations, ordering bulk reagents, and custom reagent formats, or reach out to your CST account manager.

You can also read the following paper, where researchers used PTMScan reagents in high-throughput workflows:

MRTX1719 Is an MTA-Cooperative PRMT5 Inhibitor That Exhibits Synthetic Lethality in Preclinical Models and Patients with MTAP-Deleted Cancer Engstrom et al. (Cancer Discov. 2023) | DOI: 10.1158/2159-8290.CD-23-0669 The authors used PTMScan HS Symmetric Di-Methyl Arginine Motif (sdme-RG) Kit #35985 on the Agilent AssayMAP Bravo system to interrogate patterns of symmetric dimethylarginine (SDMA) PTMs, enabling them to determine PRMT5-dependent methylation changes in response to MRTX1719 treatment in MTAP-deleted versus wild-type cancer models.

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Liquid-Handler Platforms

PTMScan HS kits are compatible with automated liquid-handler platforms. Compatible systems include the Biomek i-Series Automated Liquid Handling Workstations from Beckman Coulter and the Microlab Automated Liquid Handler line from Hamilton.

Automated liquid handling platforms generally rely on a series of steps to pipette solutions from a common reservoir to individual wells containing the PTMScan HS bead reagent, followed by mixing via aspiration, positioning the entire plate onto a magnetic module to attract the beads to the side or bottom of the well, and finally exchanging or collecting the solutions.

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Breaking the Bottleneck: Choosing the Right Proteomics Automation Strategy

PTMScan kits from CST provide flexible solutions for automating PTM enrichment across a range of robotic platforms. Whether using magnetic bead-based PTMScan HS kits for high-throughput bead-handling systems or PTMScan “classic” antibody formulations for hybrid platforms like the AssayMAP Bravo system, researchers can achieve greater reproducibility, depth, and scalability in quantitative proteomics studies.

其他资源

• To learn more about assessing reproducibility in proteomics experiments, read the blog on how to use spike-in synthetic control peptides with PTMScan.
• 网络讲座：Proteomics profiling of post-translational modifications in early drug discovery | 演讲嘉宾：Don Kirkpatrick, PhD, K48 Consulting; Lilian Phu, Greentech; and Matt Stokes, PhD, CST

Alissa Nelson, PhD, Principal Scientist in the Proteomics Group at Cell Signaling Technology, also contributed to the writing of this blog post.

SELECT REFERENCES

1. Engstrom LD, Aranda R, Waters L, et al. MRTX1719 Is an MTA-Cooperative PRMT5 Inhibitor That Exhibits Synthetic Lethality in Preclinical Models and Patients with MTAP-Deleted Cancer. Cancer Discov. 2023;13(11):2412-2431. doi:10.1158/2159-8290.CD-23-0669
2. Montoya S, Bourcier J, Noviski M, et al. Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127. Science. 2024;383(6682):eadi5798. doi:10.1126/science.adi5798
3. Crowe C, Nakasone MA, Chandler S, et al. Mechanism of degrader-targeted protein ubiquitinability. Sci Adv. 2024;10(41):eado6492. doi:10.1126/sciadv.ado6492
4. Safa-Tahar-Henni S, Páez Martinez K, Gress V, et al. Comparative small molecule screening of primary human acute leukemias, engineered human leukemia and leukemia cell lines. Leukemia. 2025;39(1):29-41. doi:10.1038/s41375-024-02400-w
5. Rivera KD, Olive ME, Bergstrom EJ, et al. Automating UbiFast for High-throughput and Multiplexed Ubiquitin Enrichment. Mol Cell Proteomics. 2021;20:100154. doi:10.1016/j.mcpro.2021.100154
6. Gritsenko MA, Tsai CF, Kim H, Liu T. Automated Immunoprecipitation Workflow for Comprehensive Acetylome Analysis. Methods Mol Biol。2024;2823:173-191. doi:10.1007/978-1-0716-3922-1_12
7. Liu Q, Adhikari E, Lester DK, et al. Androgen drives melanoma invasiveness and metastatic spread by inducing tumorigenic fucosylation. Nat Commun. 2024;15(1):1148. Published 2024 2020.02.7. doi:10.1038/s41467-024-45324-w