Abstract:

A novel method has been established based on metal element chelated tags coupled with high performance liquid chromatography-selected ion monitoring mass spectrometry (HPLC-SIM/MS). The labeling efficiency and stability of metal element chelated tags, the chromatographic retention behavior and MS behavior of the labeled peptides, the linear range and accuracy of this method were examined. The results showed that the metal element chelated tag method has high labeling efficiency and high labeling stability, and the labeled peptides with different kinds of metal tags have consistent chromatographic retention behavior. The method of metal tags coupled with HPLC-SIM/MS has high sensitivity with the limit of quantification (LOQ) up to 1 fmol. The linear range for the method was between 1 fmol to 500 fmol with R²>0.99, which means the method has a good linearity. Moreover, this method had an average recovery of 117.01%. The method was used in the absolute quantitation of a protein enolase in Thermoanaerobacter tengcongensis (TTE) with a relative standard deviation of 5.74%, which means high precision. All the results showed that this method is accurate and reliable for the absolute quantitation of proteins. This gives us an alternative for the quantitative determination of proteins in relatively simple biological samples.

Key words: absolute quantitation, high performance liquid chromatography (HPLC), metal labeling, proteins, selected ion monitoring mass spectrometry (SIM/MS)