Direct Quantitation of Intact Proteins By Multiple Ion Chromatogram Method on Q-TOF Mass Spectrometer

Significance of the topic

Accurate and precise quantification of intact proteins is critical for development and quality control of biopharmaceuticals. Monitoring protein levels across production and clinical stages ensures consistency and safety. Mass spectrometry has become a powerful tool for protein analysis but direct quantification of intact proteins remains challenging due to complex ion envelopes and matrix interferences. The multiple ion chromatogram approach on high resolution Q TOF instruments offers a potential solution for rapid and reliable measurement of protein therapeutics.

Study objectives and overview

This study aimed to evaluate a direct quantification method for intact proteins using a Shimadzu LCMS 9030 quadrupole time of flight mass spectrometer. A multiple ion chromatogram method was developed to capture the most abundant monoisotopic signals from the multiply charged ion envelope of each protein. Calibration performance, sensitivity, accuracy, precision and matrix effects were assessed for a set of model proteins across a range of molecular weights.

Used Instrumentation

• Liquid chromatography system with ProteCol G C8 column 100 mm x 2.1 mm 3 micrometre particle size column temperature 50 degrees Celsius
• Mobile phases composed of 0.1 percent trifluoroacetic acid in water and acetonitrile with a gradient from 20 to 80 percent organic at 0.3 millilitre per minute flow rate
• Shimadzu LCMS 9030 Q TOF mass spectrometer with heated electrospray interface at 300 degrees Celsius interface voltage 4 kilovolts nitrogen and zero air as nebulizing and heating gases
• Data acquisition in positive mode over 1000 to 3500 mass to charge ratio range

Methodology

Protein standards comprising ribonuclease B glycoforms insulin lysozyme and beta lactoglobulin were prepared in 0.1 percent TFA aqueous solutions and diluted over calibration ranges of 1 to 200 micrograms per millilitre. Samples were injected directly without digestion. Three highest intensity multiply charged ions were selected from the m z envelope of each protein and the corresponding monoisotopic masses were combined to form the MIC transition with a five parts per million tolerance. Calibration curves were generated in duplicate across six concentration points insulin four points and performance metrics were evaluated.

Main results and discussion

Linear calibration with correlation coefficients above 0.997 was achieved for all tested proteins within their dynamic ranges. Limits of detection ranged from 0.32 to 5.40 micrograms per millilitre and limits of quantification from 1.13 to 17.99 micrograms per millilitre. Accuracy errors remained below 20 percent while precision exhibited relative standard deviations under 15 percent. Analysis in human serum and urine matrices showed a general signal enhancement of 23 to 82 percent highlighting the importance of evaluating matrix effects in direct quantification workflows.

Benefits and practical applications

The MIC approach on high resolution Q TOF instruments enables direct rapid quantification of intact proteins without enzymatic digestion or extensive purification. The method demonstrates high sensitivity and reproducibility across proteins from 5.8 to 18.4 kilodaltons and is readily adaptable to routine biopharmaceutical analysis quality control and process monitoring.

Future trends and potential applications

Further extension to larger proteins and complex biological samples may expand the utility of MIC based quantification. Integration with automation multiplexed assays and affinity enrichment could enhance throughput and specificity. Advances in high resolution mass spectrometry and data processing will continue to improve dynamic range and matrix tolerance for intact protein measurements.

Conclusion

A simple and robust multiple ion chromatogram method on the Shimadzu LCMS 9030 Q TOF instrument provides accurate precise and sensitive quantification of intact proteins directly from solution. This strategy streamlines workflows and supports high throughput analysis in research and industrial laboratories.

Reference

• Mao Y Moore RJ Wagnon KB Pierce JT Debban KH Smith CS Dill JA Fuciarelli AF Analysis of alpha 2u globulin in rat urine and kidneys by liquid chromatography electrospray ionization mass spectrometry Chem Res Toxicol 11 1998 953 961
• Wang EH Combe PC Schug KA Multiple reaction monitoring for direct quantitation of intact proteins using a triple quadrupole mass spectrometer J Am Soc Mass Spectrom 27 2016 886 896