Strategic Use of Immunoprecipitation and LC/MS/MS for Trace-Level Protein Quantification: Myosin Light Chain 1, a Biomarker of Cardiac Necrosis
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- 作者：Michael J. Berna ; Yuejun Zhen ; David E. Watson ; John E. Hale ; Bradley L. Ackermann
- 刊名：Analytical Chemistry
- 出版年：2007
- 出版时间：June 1, 2007
- 年：2007
- 卷：79
- 期：11
- 页码：4199 - 4205
- 全文大小：137K
- 年卷期：v.79,no.11(June 1, 2007)
- ISSN：1520-6882
文摘
Myosin light chain 1 (Myl3) is a 23-kDa isoform of one ofthe subunits of myosin, a protein involved in musclecontraction. Myl3 is presently being studied as a biomarker of cardiac necrosis to predict drug-induced cardiotoxicity, and in the work presented here, an LC/MS/MSassay was developed and validated to measure Myl3 inrat serum. The key steps in this approach involvedimmunoaffinity purification of Myl3 from serum followedby on-bead digestion with trypsin to release a surrogatepeptide. This tryptic peptide was quantified using asynthetic peptide standard and a corresponding stableisotope-labeled internal standard, and the results werestoichiometrically converted to Myl3 serum concentrations. Myl3 concentrations were corrected for peptiderecovery following immunoprecipitation and digestion(85%) and showed excellent agreement with syntheticpeptide standards. Both the synthetic peptide and His-Myl3 protein were used to evaluate assay accuracy (% RE)and precision (% CV), which were measured on each of 3days. The synthetic peptide was evaluated over the rangeof 0.073-7.16 nM, while Myl3 protein QC samplesprepared in rat serum were evaluated over the range of0.13-6.62 nM. To prepare control matrix, endogenousMyl3 was immunodepleted from pooled rat serum. Peptide interday accuracy and precision did not exceed 7.6and 11.1%, and Myl3 interday accuracy and precision didnot exceed 12.9 and 13.2%, respectively. Data arepresented from the application of this assay to establisha time course in which rats demonstrated a markedincrease in Myl3 serum concentrations following administration of isoproterenol, a 
-adrenergic receptor agonistknown to induce cardiac injury. This assay is an exampleof a larger effort in our laboratory to use LC/MS/MS inconjunction with immunoaffinity techniques to evaluatecandidate biomarkers of target organ toxicity and toexpedite the development of biomarker assays for drugdevelopment.
-adrenergic receptor agonistknown to induce cardiac injury. This assay is an exampleof a larger effort in our laboratory to use LC/MS/MS inconjunction with immunoaffinity techniques to evaluatecandidate biomarkers of target organ toxicity and toexpedite the development of biomarker assays for drugdevelopment.