quality MRM methods to obtain the best sensitivity and dynamic range for peptides from a panel of putative protein biomarkers for cardiovascular disease. For candidate proteins previously detected in proteomics experiments, MRMs were designed based on peptide MS/MS spectra. For the remaining candidates, MRMs were developed either by in silico design, based on gene or protein sequences, or by targeted, direct detection in plasma using the MIDAS workflow.
The MIDAS workflow provides a straight-forward link between the genomics/proteomics biomarker discovery experiments and high throughput, quantitative MS validation, because no previous experimental data on the individual proteins of interest is required to develop an assay. This quantitative LC-MRM method profiles 53 high and medium abundant proteins in human plasma (Figure 1). Proteins down to ~1 μg/mL concentration in plasma can be reliably detected in both digested and depleted/digested human plasma, producing a dynamic range of ~5 orders of magnitude in this single method (Figure 1, inset). Peptide MRM measurements in plasma digests thus provide a robust platform for quantitative protein biomarker early stage validation.
MIDAS Workflow for Method Development:
During an MRM experiment, the triple quadrupole capability of the mass spectrometer transmits the parent ion and subsequent fragment ion with high sensitivity and selectivity. The first mass analyzer is set to transmit only the mass of the peptide parent ion (Q1 at unit resolution) into the collision cell. Only one of the sequence ions of the peptide, generated by fragmentation in the collision cell, is passed through the second mass analyzer (Q3 at unit resolution) to the detector (Figure 2).
This MRM experiment can be used as a survey scan in a MIDAS workflow to selectively detect low level peptides in complex mixtures. The detection of the pePage: All 1 2 3 4 5 Related biology technology :1
. Promegas Multiplexed Cell Viability and Apoptosis Assays performed on the PHERAstar2
. Promegas Multiplexed Luciferase Reporter and Cell Viability Assays performed on the PHERAstar3
. Mx4000 Multiplex Quantitative PCR System*,PPP4
. Improved Quantitative Selectivity of Clenbuterol in Human Urine using
High Resolution on the TSQ Quantum Mass Spectrometer5
. The LightCycler is the only System for Quantitative PCR that is as Flexible
. Quantitative Measurement of Cell Proliferation
Using the BrdU ELISA: A Comparison
Between Colorimetric and Chemiluminescent Detection7
. Quantitative end-point RT-PCR gene
expression measurement using the
Agilent 2100 Bioanalyzer and
. Detection of Genetically Modified Soybean in Processed Foods Using Real-Time Quantitative PCR with SYBR Green I Dye on the DNA Engine Opticon 2 System9
. Qualitative and Quantitative Assessment of Plasmid DNA Isolation, Rev A10
. Quantitative Analysis of Protein-DNA Associations in Vivo Using Real-Time PCR, Rev A11
. Rapid, Reproducible Real-Time Quantitative RT-PCR Using the iCycler iQ Real-Time PCR Detection System and iQ Supermix, Rev A