Automated ProteinWorks™ Sample Preparation Using Andrew+™ Pipetting Robot for Gelatin Speciation Analysis with ACQUITY™ Premier UPLC™ and Xevo™ TQ Absolute

Significance of the Topic

Gelatin is widely used in food and pharmaceutical products for its gelling properties, but porcine-derived gelatin is non-halal and restricted for certain markets. Accurate speciation analysis is essential to verify product compliance and protect consumer trust.

Objectives and Study Overview

This study aimed to enhance the efficiency and reproducibility of a three-step ProteinWorks digestion protocol for gelatin speciation by automating sample preparation with the Andrew+ pipetting robot. The resulting peptides were quantified by LC-MS/MS using an ACQUITY Premier UPLC system coupled to a Xevo TQ Absolute mass spectrometer.

Methodology and Instrumentation

The modified three-step digestion protocol reduced trypsin volume by 25 percent and was programmed in OneLab software for fully automated reagent addition, mixing, heating, and cooling. Key instrumentation included:

• Andrew+ Pipetting Robot with Peltier+ temperature control
• ACQUITY Premier UPLC HSS T3 column, 2.1 × 100 mm, 1.8 μm
• Xevo TQ Absolute tandem quadrupole mass spectrometer operating in positive ESI mode
• OneLab cloud-native protocol design and execution platform

Main Results and Discussion

Automation doubled sample preparation speed compared with manual pipetting and minimized human error. Procedural calibration curves for porcine markers in halal candy (0.025 to 1 percent) showed excellent linearity (r2 > 0.998) and accuracy (residuals < 21 percent). Method sensitivity reached an LOD of 0.01 percent and LOQ of 0.025 percent porcine gelatin. Repeatability (% RSD < 8) and reproducibility over two days demonstrated robust performance. Ten commercial candy samples were screened, confirming the method’s suitability for routine speciation analysis.

Benefits and Practical Applications of the Method

• Significantly increased throughput through automated, walk-away operation
• Reduced risk of pipetting errors and inconsistent reagent handling
• High sensitivity and reliable quantitation of species-specific peptide markers
• Scalable workflow adaptable to large sample batches in quality control laboratories

Future Trends and Potential Applications

Robotic automation platforms are expected to integrate further with laboratory information management systems for end-to-end traceability. Advances in software intelligence may enable adaptive protocols optimized in real time. The approach can extend to other proteomic speciation challenges, such as collagen sources in cosmetics or allergen detection in complex matrices.

Conclusion

This work demonstrates that automating a modified ProteinWorks digestion protocol with Andrew+ significantly improves sample preparation efficiency and reproducibility. Combined with sensitive UPLC-MS/MS analysis on the Xevo TQ Absolute, the method provides a reliable, high-throughput solution for gelatin speciation in halal compliance testing.

Reference

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2. Yin Ling Chew, Jun Xiang Lee. A complete solution for gelatin species authentication using ProteinWorks Auto-eXpress Digest Kit and Xevo TQ-XS. Waters Application Note. 2022.
3. Song E, Gao Y, Wu C, et al. Targeted proteomic assays for quantitation of proteins identified by proteogenomic analysis of ovarian cancer. Sci Data. 2017;1–13.