Protein Quantification and Quantitative Phosphorylation Analysis by the Determination of Hetero Atoms (S and P) by Means of nanoHPLC-ICPMS

It becomes easier to identify proteins via determination of peptide mass or peptide sequence by ESI-MS or MALDI-MS. On the other hand, the quantification of proteins remains a major challenge in modern proteomics. Protein quantification was performed by the determination of sulfur (S) in trypsin-digested peptides by means of nanoHPLC-ICPMS under a flow of O2 in an octopole reaction cell. Peptide concentrations were calculated as the ratio of the S concentration/the number of S atoms in the peptide/peak. Finally, protein concentration can be quantified through the peptide quantification, after taking into account tryptic digestion efficiency, via S quantification or phosphorus (P) quantification using ICPMS. This method was applied to human serum albumin and β-casein, and then the quantified protein concentrations showed a good agreement with the theoretical values, respectively. Degree of phosphorylation was also calculated as the P concentration divided by the peptide concentration. This quantitative phosphorylation analysis was applied to β-casein in cow milk.