Comparative Study of Targeted and Label-free Mass Spectrometry Methods for Protein Quantification
2013
IJsselstijn, Linda | Stoop, Marcel P. | Stingl, Christoph | Sillevis Smitt, Peter A. E. | Luider, Theo M. | Dekker, Lennard J. M.
We compared data acquired on an LTQ-Orbitrap MS used in a typical shotgun proteomics setting (optimized for protein identification) with data from a quadrupole ion trap MS operated in the MRM mode. Six relative abundant proteins were quantified in identical sets of serum and CSF samples by the following methods: a qual/quant method with and without use of internal standards and a quantitative method (MRM with use of internal standards). Comparison of these methods with an antibody-based method in CSF samples showed good linearity for both methods (R² of 0.961 and 0.971 for the qual/quant method with use of internal standards and the quantitative method, respectively). Besides its better linearity, the quantitative method was also more reproducible with lower CVs for all samples. Next to these comparisons we also explored why a qual/quant approach had typically a lower reproducibility compared to MRM analyses. We observed that modified peptides, or peptides with a cysteine or a methionine, yielded a significant increase in CV. Furthermore, a positive correlation was found between the length of the peptide and the CV. We conclude that qual/quant is an alternative for the quantification of abundant proteins and that the use of internal standards in qual/quant could be advantageous. Furthermore, the ongoing development in MS techniques increases the possibilities of qual/quant in protein quantification.
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