Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Novel LC MS-based strategies to detect small changes in the plasma proteome for metaproteomics studies (#501)

Jean-Jacques Dunyach 1 , Scott Peterman 1 , David Sarracino 1 , Jae Choi 1 , Michael Belford 1 , Jesse Canterbury 1 , Graeme McAlister 1 , Jacob Schwartz 1 , Aaron Robitaille 1 , Romain Huguet 1 , Vlad Zabrouskov 1
  1. Thermo Fisher Scientific, San Jose, CALIFORNIA, United States

New analytical strategies continue to be developed to expand the depth of plasma proteome profiling for putative biomarker discoveries. The enormous dynamic range and vast donor variability of non-depleted plasma presents significant challenges to low-level protein expression level perturbations needed for metaproteomics.  To address this, we have developed a method combining multiplexed TMT11 labeling and UHPLC separations with a new Tribrid mass spectrometer equipped with a FAIMS interface to perform SPS-MS3 with Real-Time search.  A common plasma stock is used to create the reference standard and 4 “donor” samples each with a different amount of E. coli digest spiked prior to labeling with the different TMT11 tags. The plasma standard is labeled with the TMT0 tag to create an MS-level normalization. The amount of E. coli digest spiked into each 1 µg plasma digest sample increases from 50 to 500 ng prior to labeling.  A 1:1 mixture of the standard and donor sample is analyzed with and without FAIMS as well as with and without the Real-Time Search routine. The initial results demonstrate that incorporating stepped FAIMS CV settings increases the loading capacity which is critical to handling the plasma standard spiked with the TMT11 samples.  Additionally, the stepped FAIMS CV settings within one experiment increases the dynamic range over 3-fold increasing the number of E. coli peptides and proteins accurately measured per donor sample.  Lastly, incorporation of the Real-time search algorithm further increased E. coli peptide and protein detection as compared to both MS2 and traditional SPS-MS3 routines.