Introduction
Targeted LC-MS assays are increasingly applied in the post-discovery proteomics area with an emphasis on validation. A novel DIA mode of operation, which provides both qualitative and quantitative information, has been developed for a tandem quadrupole/oa-time-of-flight (ToF) MS and applied to targeted proteomics experiments using transition extraction lists and compound library based approaches.
Method
NanoLC was performed with a Waters Nanoacquity UPLC equipped with a 75micron x 150mm column. The m/z isolation range of the quadrupole was continuously scanned with MS data acquired up to a frequency of 400 spectra/s. Alternate MS scans comprised precursor and CID product ions. The resulting 2D data, m/z (ToF) vs. m/z (quadrupole) were processed using Progenesis QIP and Skyline informatics.
Results
Quantitative proof-of-principle data were acquired by serially diluting a four protein digest mixture into a proteolytic E.coli digest and quantitative information obtained by using transition extraction lists. Analysis of the data indicates that scanning quad DIA enables over an order of magnitude more specificity than a static quad operated with the same resolution and it was found that a quadrupole transmission window of approx 20 - 30 Da provided optimum protein identifications.
Aliquots of control, diabetic and obese human plasma samples were digested with trypsin overnight. 2DMS data were collected and differentially expressed peptides and hence proteins across the three conditions were quantified. Results show that peptides from apolipoproteins exhibited the most significant changes in good agreement with expected changes in relation to disease and/or phenotype.
Novel Aspect
Targeted quantitation in proteomics using a novel precursor quadrupole scanning based DIA method