Strategic use of immunoprecipitation and LC/MS/MS for trace-level protein quantification: myosin light chain 1, a biomarker of cardiac necrosis.
Myosin light chain 1 (Myl3) is a 23-kDa isoform of one of the subunits of myosin, a protein involved in muscle contraction. 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/MS assay was developed and validated to measure Myl3 in rat serum. The key steps in this approach involved immunoaffinity purification of Myl3 from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified using a synthetic peptide standard and a corresponding stable isotope-labeled internal standard, and the results were stoichiometrically converted to Myl3 serum concentrations. Myl3 concentrations were corrected for peptide recovery following immunoprecipitation and digestion (85%) and showed excellent agreement with synthetic peptide 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 3 days. The synthetic peptide was evaluated over the range of 0.073-7.16 nM, while Myl3 protein QC samples prepared in rat serum were evaluated over the range of 0.13-6.62 nM. To prepare control matrix, endogenous Myl3 was immunodepleted from pooled rat serum. Peptide interday accuracy and precision did not exceed 7.6 and 11.1%, and Myl3 interday accuracy and precision did not exceed 12.9 and 13.2%, respectively. Data are presented from the application of this assay to establish a time course in which rats demonstrated a marked increase in Myl3 serum concentrations following administration of isoproterenol, a beta-adrenergic receptor agonist known to induce cardiac injury. This assay is an example of a larger effort in our laboratory to use LC/MS/MS in conjunction with immunoaffinity techniques to evaluate candidate biomarkers of target organ toxicity and to expedite the development of biomarker assays for drug development.
Berna
Michael J
MJ
Lilly Research Laboratories, Eli Lilly and Company, Greenfield, Indiana 46140, USA. mberna@lilly.com
Zhen
Yuejun
Y
Watson
David E
DE
Hale
John E
JE
Ackermann
Bradley L
BL
D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D006331:Heart Diseases / Q000378:metabolism*; D047468:Immunoprecipitation / Q000379:methods*; D007545:Isoproterenol; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000737:chemistry* / Q000378:metabolism*; D009336:Necrosis / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry / Q000379:methods*
D015415:Biomarkers; D018994:Myosin Light Chains; C076659:myosin light chain I; D007545:Isoproterenol