Developing the Research to Routine Workflows with FAIMS: Automating Large-Scale SRM Method Creation for Routine Plasma Proteomics Screening
Kerry Hassell, Debadeep Bhattacharyya, Mary Blackburn, Michael Belford, Michael Volny, Scott Peterman, Romain Huguet Thermo Fisher Scientific, San Jose, CA
Warning: Undefined variable $headshot in /var/www/html/view_abstract/view_abstract_in_program.php on line 704
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Introduction:
Highly multiplexed protein panels are developed to enable routine sample screening while maintaining high throughput. The challenge to creating an analytically robust SRM method is determining which peptides to select per protein and creating the resulting SRM table for confident data acquisition. Each protein added to the target list increases total SRM count by 9 quickly causing acquisition challenges on triple quadrupole mass spectrometers as most proteotypic peptides cluster into small hydrophobicity groups. To increase the selectivity space, we have incorporated a field asymmetric waveform ion mobility spectrometry (FAIMS) interface for both profiling and screening to increase the selectivity metrics for an SRM method monitoring over 300 plasma proteins in 60 minutes.
Experiment:
A stock solution of tryptic plasma digest was used for all experiments. Plasma profiling was performed using an Orbitrap tribrid mass spectrometer with a FAIMS interface. A total of 2 µg of the plasma digest was injected and analyzed using a single compensation voltage (CV) setting by standard DDA methods and repeated for eight different CV settings. Each RAW file was processed to create a data matrix of proteins and peptides, retention time, CV, and precursor and product ion distribution profiles. A routine was created to construct a scheduled SRM table for the top 300 plasma proteins using over 2500 SRM transitions. The SRM table was imported into a triple quadrupole mass spectrometer with the FAIMS interface and evaluated for analytical performance.
Preliminary Data:
The discovery method was used to fully characterize the non-depleted plasma digest. Replicate sample injections using single CV settings significantly increases the protein coverage from 310 proteins without FAIMS to over 500 proteins with FAIMS. In addition, the resulting data is used to create a four-dimensional library that consists of the protein and corresponding peptides, and for each peptide, the measured retention time, CV setting, and optimal precursor m/z value and product ion distribution is recorded. The addition of FAIMS enhances the selectivity and sensitivity of peptides increasing the number of available peptides per targeted protein resulting in more options to be considered in creating the scheduled SRM table.
For the different protein groups targeted, the optimal peptides were selected based on relative response in the discovery method, but more importantly on the retention time and CV setting as the two values were used to create the final SRM table. Peptides were groups into overlapping retention time and CV bins to maximize duty cycle while maintaining analytical performance. Initial results for the 100 and 200 targeted protein tables result in over 85% and 80% for the peptides maintaining acceptable quantitation ion ratio calculations. The peptide selection process was reevaluated prior to moving on to the 300 targeted protein list.
= Discovery stage.
= Translation stage.
= Clinically available.
