Title: PROTEIN QUANTIFICATION WITH ELEMENTAL TAGS USING ICP-MS
Blakely Adair, Eric Kilpatrick, Guillaume Ballihaut, Clay Davis, Greg Turk, Stephen Wise, Stephen Long, and Steven Christopher
The increased interest and understanding of protein structure and function has resulted in a need for better protein quantification. Crucial steps toward improving protein quantification include accurate, sensitive, and specific analytical methodologies. The goal is to develop and validate methods that complement biomolecular mass spectrometry and bioassays but can be used for absolute protein content determinations, such as quantification of proteins in NIST SRMs. One approach utilizes bifunctional lanthanide ligands to tag proteins with an elemental label that can be detected with ICP-MS.
C-reactive protein (CRP) was the model protein. This stable protein has been studied extensively; therefore, standards and monoclonal antibodies (MAb) were available. We used 1-[p-(3,5-dichlorotriazinyl)benzyl] diethylenetriamine-N1,N1,N2,N3,N3-pentaacetic acid chelated to Eu (DTPA-Eu) as a bifunctional ligand to tag CRP. Paramagnetic beads with MAb were tested in preparation for CRP isolation from serum to determine the binding affinity of the CRP-DTPA-Eu complex. After labeling, size exclusion chromatography (SEC) was linked to UV and ICP-MS to separate labeled proteins for detection. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) was used to identify and verify CRP binding with DTPA-Eu and MAb.
MALDI-MS spectra had a signal for CRP-DTPA-Eu at 800 m/z units greater than CRP which verified that DTPA-Eu bound to CRP. Europium peaks on ICP-MS were seen at retention times that matched CRP as detected by UV, further verifying that CRP did bind to DTPA-Eu. The Eu signal measured by ICP-MS for a CRP concentration of 0.8 µg/ml was ten times the baseline signal and well within the range of ELISA techniques for CRP determination. MALDI-MS and SDS-PAGE gels demonstrated that the CRP-DTPA-Eu complex did bind to the MAb in affinity purification by comparison with CRP. Method development will continue by assessing concentration limitations of CRP binding to DTPA-Eu, CRP elution conditions from MAb, and matrix interferences from serum.
NIST Analytical Chemistry Division, Hollings Marine Lab Charleston, SC 29412
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Mentor’s Name: Steven Christopher
Tel: 843 762-8856
Fax: 843 762-8742
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