ptide by MRM drives the acquisition of MS/MS to confirm the peptide sequence and thus definitively identity of the detected peptide (Figure 3). Because of the high specificity and sensitivity of MRM, these experiments possess better signal/noise than full scan MS signals and can be used to dig deeper into a sample. The hybrid triple quadrupole linear ion trap technology of the 4000 Q TRAP system is the only platform that enables this combination of specific MRM detection with high sensitivity ion trap MS/MS. The peptide MRM transitions can be determined either experimentally from previously obtained proteomics data or in silico by using the MIDAS workflow software designer. From the protein sequence, theoretical tryptic peptides are determined and the fragmentation pattern of the peptide by MS/MS is predicted (Figure 4). This strategy is key to developing MRMs with good S/N to low abundance proteins that are often not observed in regular LC/MS/MS methods. In addition, putative biomarkers identified from a genomics based discovery platform can also be included in this validation strategy as all that is required is the sequence of the protein. No peptide or protein standards are required for method development.

High Quantitative Reproducibility:

A key component to the early and late stage validation of biomarkers in any body fluid is the ability to prepare and analyze many samples in parallel in a highly reproducible manner. The reproducibility of the LC-MRM method was assessed by measuring 10 LC-MRM replicates on the same sample. With no internal standard correction, the reproducibility of the peak areas was better than 10% for the majority of the MRM transitions (Figure 5, right). The average %CV for all MRM peaks was 8.6% for depleted plasma (loading equivalent of 10 nL of plasma) and 11.7% for undepleted plasma (loading 1 nL equivalent).

The reproducibility of both the depletion and dig
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