Identification of novelin vitroprotein kinase A phosphorylation sites on recombinant non-muscle myosin light chain kinase: nano-liquid chromatography tandem mass spectrometry methodology

2009 ◽  
Vol 5 (4) ◽  
pp. 242-253 ◽  
Author(s):  
Jing Zhao ◽  
Sara M. Camp ◽  
Eddie T. Chiang ◽  
Alexander B. Schilling ◽  
Steven M. Dudek ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Tandem Mass ◽  
Phosphorylation Sites ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Nano Liquid Chromatography ◽  
Muscle Myosin ◽  
Kinase A

scholarly journals Liquid Chromatography-Tandem Mass Spectrometry for Detecting Myosin Light Chain 3 in Dry-Aged Beef

Separations ◽  
2021 ◽  
Vol 8 (11) ◽  
pp. 219
Author(s):  
Heeyoung Lee ◽  
Yoonji Heo ◽  
Jong-Chan Kim ◽  
You-Shin Shim
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Western Blotting ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Muscle Protein ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Liquid chromatography-tandem mass spectrometry (LC/MS/MS) is a more accurate technique for detecting proteins than electrophoresis-based methods such as western blotting. Because of its convenience, western blotting is commonly used for protein analysis in beef. We developed a method for detecting myosin light chain 3 (myl3) in beef samples, particularly dry-aged beef, using LC/MS/MS for quality testing. Musculus longissimus dorsi of Holstein was aged for 0, 2, 4, 5, 9, 11, 17, 20, and 24 weeks and used to measure the myl3 concentration. Because of the high molecular weight of myl3, the limitations of LC/MS/MS were overcome by implementing immunoprecipitation and digestion steps. Ultimately, a tryptic fragment of myl3 (13-mer), generated using immunoprecipitation and digestion by a biotinylated antibody, was detected using LC-MS/MS in positive ion mode through multiple reaction monitoring and analyte separation on a C18 column. Our method showed limits of detection and quantification of less than 0.3 and 0.8 μg/kg, respectively. However, differences in the myl3 concentrations according to the aging time were not significant (p > 0.05). After 12 weeks, myl3 disappeared in tested all samples, thus our analytical method can be used for accurate measurement of muscle protein in beef samples.

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