title: Comparative study of targeted and label-free mass spectrometry methods for protein quantification.
abstract: 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 (R(2) 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.
authors: Department of Neurology, Erasmus Medical Center , Rotterdam, The Netherlands.
authors: Linda
authors: L
authors: IJsselstijn
authors: Marcel P
authors: MP
authors: Stoop
authors: Christoph
authors: C
authors: Stingl
authors: Peter A E
authors: PA
authors: Sillevis Smitt
authors: Theo M
authors: TM
authors: Luider
authors: Lennard J M
authors: LJ
authors: Dekker
mesh_terms: D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis; D002555:Cerebrospinal Fluid / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results
chemical_list: D001798:Blood Proteins; D011506:Proteins