Simple and sensitive quantitation of large therapeutic proteins in plasma in 90 minutes

Significance of the topic

Large therapeutic proteins, including monoclonal antibodies, represent a growing class of biopharmaceuticals with crucial roles in disease treatment. Reliable quantitation in biological matrices such as plasma is vital for pharmacokinetic studies, dose optimization, and safety monitoring. Traditional LC-MS/MS workflows often require extensive sample preparation, long digestion times, and complex denaturation steps, limiting throughput and reproducibility.

Objectives and study overview

This application note demonstrates a streamlined workflow for quantifying human IgG in mouse plasma within 90 minutes. The goals are to simplify sample preparation, shorten digestion time compared to conventional methods (up to 24 hours), and achieve sensitive, reproducible results across a wide concentration range without the need for peptide enrichment.

Methodology

A one-step, heat-activated digestion protocol was employed using a thermally stable trypsin formulation. Five microliters of plasma were combined with buffer and enzyme in a 96-well low-adsorption plate, sealed, and incubated at 70 °C for 90 minutes. After cooling, samples were acidified with trifluoroacetic acid and diluted prior to injection. Two signature peptides, VVSVLTVLHQDWLNGK (m/z 603.4>805.4) and TTPPVLDSDGSFFLYSK (m/z 937.7>836.5), were monitored by selected reaction monitoring on an ion-trap mass spectrometer.

Used instrumentation

• Thermo Scientific UltiMate 3000 Rapid Separation Dual UHPLC system with degasser, rapid-separation pump, thermostatted autosampler, and column compartment
• Accucore C18 column, 2.1×50 mm, 2.6 µm
• Thermo Scientific Velos Pro ion-trap mass spectrometer with HESI source

Results and Discussion

Calibration curves for both peptides were linear over 0.5–500 µg/mL, with correlation coefficients above 0.9997. The lower limit of quantitation at 0.5 µg/mL displayed coefficient of variation around 10% for VVSVLTVLHQDWLNGK and 11.6% for TTPPVLDSDGSFFLYSK. Upper limit precision improved to under 5%. Rapid thermal digestion enhanced peptide yield and signal intensity by up to 800-fold relative to lower temperatures, overcoming incomplete proteolysis observed in conventional protocols.

Benefits and practical applications

• Fast turnaround: complete protein digestion and analysis in 90 minutes versus up to 24 hours
• High reproducibility: single-step workflow reduces variability and manual handling
• Cost efficiency: lower enzyme consumption due to thermally enhanced activity
• No enrichment required: direct quantitation in plasma simplifies sample processing

Future trends and potential applications

Emerging directions include adapting this high-throughput digestion approach to multiplexed quantitation of diverse protein therapeutics, integrating automation for large-scale studies, and combining thermal digestion with immunoaffinity capture for ultra-low abundance targets. Further optimization may enable intact protein analysis and deeper peptide mapping for structural studies.

Conclusion

The SMART Digest Soluble kit and thermal digestion strategy deliver a rapid, sensitive, and reproducible LC-MS/MS workflow for large protein quantitation in plasma. By reducing sample preparation time and complexity, this method supports high-throughput bioanalysis in drug development and clinical research.

References

1. Becker JO, Hoofnagle A Replacing Immunoassays with Tryptic Digestion Peptide Immunoaffinity Enrichment and LC-MS/MS Bioanalysis 2012 4(3) 281–290
2. Keil-Dlouha V et al Proteolytic Activity of Pseudotrypsin FEBS Lett 1971 16(4) 291–295
3. van den Broek I et al Evaluation of Interspecimen Trypsin Digestion Efficiency Prior to Multiple Reaction Monitoring J Proteome Res 2013 12 5760–5774
4. Thermo Fisher Scientific SMART Digest Peptide Mapping and Quantitation Compendium 2018 AN-21878