Automation of sample preparation steps in a cPILOT quantitative proteomics workflow
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1
Vanderbilt University, United States
To facilitate disease understanding and biomarker discovery, high throughput quantitative proteomics workflows are necessary to identify thousands of proteins across large cohorts of samples. Additionally, it is beneficial to have workflows that can be completed rapidly, easily, and in a cost-effective manner. Our laboratory’s cPILOT workflow is one such example of a high throughput quantitative proteomics workflow. We have successfully demonstrated with this quantitation scheme the ability to multiplex up to 22 or 24 samples with TMT and DiLeu isobaric tags, respectively, in a single experiment. While we reduce total MS acquisition times considerably from label-free approaches and save costs on TMT reagents, the major bottlenecks in the approach lie in the sample preparation steps as there are numerous manual handling steps that are labor intensive, time consuming, and prone to sample loss and quantitative error. Thus, we are working to address these bottlenecks by incorporating sample automation into the cPILOT workflow to increase workflow efficiency, reproducibility, and quantitative accuracy. In the present study we aim to automate our novel cPILOT workflow by moving the sample preparation steps to 96-384 well-plate format in order to process hundreds of samples in parallel. To start with the automation of the sample preparation the manual de-salting step was transferred to a vacuum manifold which utilizes regulated pressure for efficient buffer exchanges between plates. Peptide recovery using the online automated desalting process will be compared to the on-cartridge desalting. To automate the entire workflow, reduced, alkylated and denatured proteins are trapped on a 96-well PES membrane plate and on-plate proteolysis performed. Eluted peptides are tagged using the cPILOT approach in a well-plate and pooled into a single sample that can be de-salted and subjected to nanoflow LC-MS, MS/MS, and MS3 analysis on various Orbitrap MS instruments. This presentation will discuss our findings on optimal experimental conditions and figures of merit such as quantitative error, inter sample variability, workflow efficiency when comparing the manual approach to the automated cPILOT platform.
Keywords:
sample preparation,
Enhanced multiplexing,
cPILOT,
Quantitative Proteomics,
TMT tags,
Dimethylation labeling
Conference:
National Organization for the Professional Advancement of Black Chemists and Chemical Engineers (NOBCChE) 45th Annual Conference , Orlando, Florida, United States, 17 Sep - 20 Sep, 2018.
Presentation Type:
Oral Presentation
Topic:
Analytical Chemsitry
Citation:
Arul
AB,
Amin
B and
Robinson
RA
(2019). Automation of sample preparation steps in a cPILOT quantitative proteomics workflow.
Front. Chem.
Conference Abstract:
National Organization for the Professional Advancement of Black Chemists and Chemical Engineers (NOBCChE) 45th Annual Conference .
doi: 10.3389/conf.fchem.2018.01.00029
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Received:
09 Oct 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Prof. Rena A Robinson, Vanderbilt University, Nashville, United States, rena.as.robinson@vanderbilt.edu