title: Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer.
abstract: Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 μg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.
authors.affiliation: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, 76019, USA.
authors.forename: Evelyn H
authors.initials: EH
authors.lastname: Wang
authors.affiliation: Shimadzu Scientific Instruments, Inc., Columbia, MD, 21046, USA.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Combe
authors.affiliation: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, 76019, USA. kschug@uta.edu.
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Schug
mesh_terms: D000818:Animals; D002138:Calibration; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D006736:Horses; D057230:Limit of Detection; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D015203:Reproducibility of Results
chemical_list: D011506:Proteins
keywords: Intact protein quantitation; Intact protein separation; Matrix effects; Prostate-specific antigen; Top-down protein analysis