MSACL 2018 US Abstract

Topic: Proteomics

Top-Down Quantitative Determination of Intact Proteins in Serum by Multi-Dimensional LC/QTOF Mass Spectrometry

Jack Henion (Presenter)
Q2 Solutions

Bio: Professor Jack Henion is Emeritus Professor of Toxicology at Cornell University where he was a member of the College of Veterinary Medicine commencing in 1976. Dr. Henion was co-founder of Advion BioSciences in 1993 and is now CSO of Advion, Inc. and VP R&D of Q2 Solutions, a Joint Venture between Quintiles and Quest. Professor Henion has received three Doctor Honoris Causa (Honorary Doctorate) degrees in recognition of his international reputation in modern analytical techniques. During his tenure at Cornell Professor Henion conducted research and explored applications in many areas of liquid chromatography/mass spectrometry (LC/MS) employing atmospheric pressure ionization (API) sources. Professor Henion has published over 225 peer reviewed papers in the scientific literature, and trained nearly 100 students, post-doctoral scientists, and trainees.

Authorship: Jack Henion and Liyun Zhang
Q2 Solutions, Inc., 19 Brown Rd, Ithaca, NY 14850.

Short Abstract

A top-down LC/MS/MS method has been developed for the quantitative determination of an intact model protein (SigmaMab) in mouse plasma. Both the intact denatured protein and its intact native form have been studied with the goal of quantifying this protein using its commercially available stable isotope internal standard, SILUMab. SEC, HIC and weak cation exchange chromatography was coupled in an automated multi-dimensional manner to a QTOF MS system. A linear dynamic range from 0.2 to 20 ug/mL plasma was accomplished for this protein in its denatured form. Results of another biotherapeutic protein from incurred monkey serum will also be reported.

Long Abstract


The quantitative determination of proteins and biotherapeutics in biological samples by LC/MS/MS has been popularized by the ‘bottom-up’ approach. This approach uses well characterized enzymatic digests which produce a mixture of peptides which made up the protein. A unique (signature or surrogate) peptide is then chosen as ‘representative’ of the protein and quantified by reversed-phase LC/MS/MS operated in the selected reaction monitoring (SRM) mode of operation [1]. Although this approach provides good sensitivity and selectivity for proteins isolated from biological samples, it has the potential to miss quantifying important protein features including post-translational modifications, disulfide linkages, oxidation or reduction of selected amino acids, etc. which may be remote from the selected surrogate peptide.

A potentially more satisfying approach is to quantify directly the intact protein. Although the so-called ‘top-down’ proteomics approach has been around for nearly 20 years, this approach has been confined to qualitative studies of proteins; not the quantitative determination of proteins. Recently there has been some developing interest in the top-down bioanalysis of large molecules including proteins [2]. This is a bit of a daunting task since it challenges our notion of current LC/MS practices. ‘Handling’ a large molecule both chromatographically and mass spectrometrically requires a different approach than our customary LC/MS/MS practices. If, ideally, we want to maintain the native conformation of the protein during sample preparation, chromatography and mass spectrometry, we will need to employ different practices to achieve success.

This report details a sample preparation strategy coupled with an automated multi-dimensional LC/QTOF MS analytical system for the top-down quantitative determination of a model protein with a molecular weight of 154 KDa (SigmaMab). A commercially available stable isotope internal standard is available so this was used to develop a method for its determination in fortified mouse plasma samples over a range from 0.2 to 20 ug/mL plasma. Following this another biotherapeutic protein with a similar molecular weight isolated from incurred monkey serum was studied to demonstrate the potential of this approach.


Commercially available SigmaMab and its stable isotope internal standard, SiluMab, (SIL) were purchased from Millipore Sigma. Measured levels of these standards were fortified from 0.2 to 20 ug/mL in mouse plasma. Sample preparation was performed where Mouse plasma (40 uL) was diluted 10x with 20 mM PBS, 0.1% BSA, 0.01% Tween-20. SigmaMab was spiked into mouse plasma from 20 to 0.2 ug/mL with separate QCs at 16, 8, 1.6, 0.2 ug/mL. Anti-human IgG Fc-Biotinylated capture antibody (12.9 uL, 3.1 mg/mL, Millipore Sigma) was added to each sample (antibody/protein ratio=5:1). The plate was shaken overnight at 10 0C. Streptavidin T1 beads (0.2 ml, 10 mg/mL, Thermo Fisher) were added to each well (200 ug antibody per mL beads). The solution mixture was incubated at room temperature for 1 hour. The beads were washed by 200 uL CHAPS buffer, 200 uL 0.1% BSA in PBS buffer, 200 uL water subsequently. After washing, 0.1% formic acid (50 uL x 3) was added to elute the analyte off the beads. The eluents were collected in a clean 96-well plate and ready for LC-QTOF MS analysis.

The chromatography system was composed of 2D multi-dimensional LC column set up. The first dimension was a SEC column: PolyHydroxyEthyl A (Poly LC), 100 x 4.6 mm, 3 um, 500 Å maintained at a flow of 0.2 mL/min, 90:10: 0.1 water/acetonitrile/formic acid. The second dimension was an RP: PLRP-S (Agilent), 150 x 2.1 mm, 8 um, 1000 Å maintained at a flow of 0.2 mL/min, MPA: 0.1% FA water; MPB: 0.1% FA acetonitrile with a gradient of 10% B for 3 min, 3-4 min 10%B-30%B, 4-10 min 30%-35%B.

The mass spectrometer was a 6545 QTOF system from Agilent technologies.


Automated multi-dimensional LC/QTOF MS bioanalysis of the above-described fortified plasma samples containing standards and QC’s across a concentration range from 0.2 to 20 ug/mL produced acceptable quantitative results. Two approaches were used for calculating the response ratios between the fortified standards and its stable isotope internal standard: one approach summed the four most abundant charge states in the multiply-charged ion envelope. This approach produced a non-linear response at the higher concentration levels. A much more linear calibration curve was obtained by employing the Agilent deconvolution software to form a single ‘zero charge state’ as a result of the deconvolution. Under these conditions this approach appeared to be preferred.

Following a relatively successful study of this target protein in fortified samples, an example of an incurred protein study was desired. A potential commercial biotherapeutic from a major pharmaceutical company provided samples of incurred monkey serum containing its biotherapeutic protein candidate from an administration study in the monkey. Results from the bioanalysis of these samples were obtained in a similar manner and will be described in this report.

Conclusions & Discussion

From these preliminary studies, it appears possible to obtain bioanalytical results from intact proteins isolated from biological samples using multi-dimensional LC/QTOF MS techniques. We and others have now reported the top-down bioanalysis of intact denatured proteins, but our major goal is to achieve similar success with intact native proteins. Although much more challenging and currently with higher LLOQ’s due to the reduced charge state distributions and the reduced response in sub-optimal electrospray solvents, the potential benefits of more inclusive information from an intact protein via this top-down approach makes the effort worth exploring further.

References & Acknowledgements:

1. Dubois, M., Fenaille, F., Clement, G., Lechmann, M., Tabet, J. C., Ezan, E., Becher, F. Immunopurification and mass spectrometric quantification of the active form of a chimeric therapeutic antibody in human serum. Analytical chemistry 2008, 80(5), 1737-1745.

2. Kang, L., Camacho, R. C., Li, W., D’Aquino, K., You, S., Chuo, V., Weng, N., Jian, W. Simultaneous Catabolite Identification and Quantitation of Large Therapeutic Protein at the Intact Level by Immunoaffinity Capture Liquid Chromatography–High-Resolution Mass Spectrometry. Analytical Chemistry 2017, 89(11), 6065-6075.


SIO2 Medical Products, Inc.

Agilent, Inc. (CA, USA)

Financial Disclosure

GrantsyesSiO2 Medical Products
Board MemberyesSMRTL: Sports Medicine Research and Testing Lab

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above: