A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Abstract

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow 1 . Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as β-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids

“…All blood biofluids underwent protein denaturation, reduction, alkylation, digestion using the Beckman i7 automated workstation (BeckmanCoulter) programed for uniform mixing as previously described at a controlled temperature and modified with online automated desalting. 13 Proteins were denatured in a solution of 35% v/v 2-2-2 Trifluoroethanol (TFE, Sigma), 40 mM Dithiothreitol (DTT, Sigma) and dissolved in 50 mM NH 4 CO 3 (Sigma). The sample was denatured for 1 hr at 60 °C.…”

Progress Identifying and Analyzing the Human Proteome: 2021 Metrics from the HUPO Human Proteome Project

“…The Van Eyk group at Cedars-Sinai has reported a refined protocol and instructions for automated programmable plasma protein digestion in 96-well plate format, which improved the accuracy, precision, and reproducibility of targeted and discovery proteomics assays for clinical implementation. 59 The Kirk group at Loyola University used ubiquitination proteomics to reveal a new link between the BAG3 protein and myofilament turnover in the setting of heart failure. 60 The Ge group at University of Wisconsin made advances with top-down proteomics to characterize proteoform permutations and discover that different genetic variants associated with hypertrophic cardiomyopathy funnel into common patterns of altered sarcomeric proteoforms that are predictive of disease phenotypes and may represent common intervention targets.…”

Proteomics: A Tool to Study Platelet Function

“…With protein low-binding tubing material, the method could also be applicable for proteomic sample preparation. The additional controls, ensuring complete platelet lysis and digestion [ 117 , 118 ], minimal loss of peptide during sample preparation, and automation for the analysis of hundreds or thousands of samples should be established for high-throughput studies [ 119 ]. Finally, quality control systems of mass spectrometry-based proteomics data acquisition should be introduced in the labs dealing with platelets’ biomedical and translational applications, e.g., a cloud-based quality control system [ 120 ] or web-based applications [ 121 , 122 ].…”