(M1330-06-36) Determination of Microdystrophin (h-µD5) via Signature Peptide Analysis in Muscle Tissue by LC-MS/MS

Purpose: Bicinchoninic acid (BCA) assay and stable isotope standards and capture by anti-peptide antibodies (SISCAPA) methods combined with liquid chromatography-mass spectrometry (LC-MS/MS) was developed to detect Microdystrophin (h-µD5) using reference standard via selected signature peptides A and B.

Methods: These selected signature peptides were chosen to represent proteins Microdystrophin (h-µD5) in muscle tissue. The BCA protein assay was used for quantitation of total protein in a homogenate tissue sample and normalize the protein concentration to 3 mg/mL. Selected signature peptides A and B were immunocaptured via antibodies raised against each peptide and immunoprecipitated by SISCAPA following with a Sciex API 6500+ LC-MS-MS analysis. SISCAPA method for specific antibody-based capture and precipitation of individual peptides from a homogenate tissue was developed using a simplified magnetic bead protocol.

Results: This innovative analytical approach was validated and found to be accurate and precise. The range of quantitation was 500 pM to 100,000 pM for both peptides A and B based on the analysis of 50.0 µL of homogenized muscle tissue.

Conclusion: SISCAPA technology combines the best features of mass spectrometry and immunoassays to purify, isolate and measure selected signature peptides accurately and efficiently in complex sample matrix.

Antibody Mounting to magnetic beads

Bicinchoninic acid (BCA) Assay for Total Protein Content at 3mg/mL

SISCAPA Workflow With LC-MS/MS (Via immunocapture and Immunoprecipitation)