ac300584y_si_001.xls (623 kB)
An Assessment of Peptide Enrichment Methods Employing mTRAQ Quantification Approaches
dataset
posted on 2012-07-03, 00:00 authored by David
N. Potier, John R. Griffiths, Richard D. Unwin, Michael
J. Walker, Emma Carrick, Andrew J. K. Willamson, Anthony D. WhettonThe human plasma peptidome has potential in biomarker
discovery
not least because the plasma proteome is a challenging matrix due
to its complexity and dynamic range. However, methods to significantly
reduce the amount of protein present in plasma while retaining the
less abundant peptides present in plasma samples has been a major
issue. Here, we present a novel strategy which has been employed to
assess the effectiveness of removing interfering proteins while retaining
peptides of interest. To monitor peptide retention, a spiked in digested
protein, in this case a synthetic QconCAT protein, was employed. This
enabled a variety of target analytes (peptides) to be monitored for
their retention in liquid phase, providing a broader picture of peptide
loss from each method assessed. The incorporation of mTRAQ labeling
allowed the presence of each peptide to be monitored, and accurate
peptide losses to be determined in a Selected Reaction Monitoring
(SRM) assay, thus, enabling an objective semiquantitative conclusion
to be drawn regarding the suitability of each method for protein removal
and peptide retention. We also assessed a range of methods for retaining
nontryptic peptides in a plasma peptidomics workflow. From these data,
we determined an optimal workflow for removing intact protein, while
retaining peptides for MS-based analyses.