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Multiplexed Quantitative Peptide Assays for Protein Biomarkers of Cardiovascular Disease in Human Plasma

estion step of the plasma preparation was explored by taking one sample, splitting it in two and performing parallel depletions, followed by a further split of each to perform parallel digestions. All samples were then assayed and correlated (Figure 5, left). Initial results suggest that these types of sample preparation techniques can be performed in a highly reproducible manner.

MIDAS TRAQ System optimized for the MIDAS Workflow

To enable the MIDAS workflow at optimum performance levels, Applied Biosystems/MDS SCIEX has introduced the MIDAS TRAQ LC/MS/MS system. The system combines the reproducibility and ease-of-use of the new Tempo nano MDLC system with the high sensitivity and dynamic range of the 4000 Q TRAP hybrid triple quadrupole linear ion trap system. The system is also fully compatible with iTRAQ reagents for protein biomarker discovery.

Conclusions

A single period multiplexed MRM assay consisting of 137 MRM experiments representing 53 plasma proteins (including 38 cardiovascular disease biomarkers) has been developed and tested in digested human plasma.2 Very good reproducibility between replicates and across sample preparations was obtained, highlighting the potential of this method for higher throughput quantitative biomarker early stage validation.

A key strategy to the design of these methods was the use of the MIDAS workflow for developing the MRM transitions to previously undetected proteins and improving MRM selection and sensitivity to others. With this straightforward strategy, proteins in the top 5 orders of the protein abundance curve in human plasma can be quantitatively profiled in a high throughput manner with minimal sample and minimal sample preparation.

This same strategy can be applied to proteins of lower abundance, using additional upfront sample preparation.

The MIDAS workflow prov
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