John Wilson (Presenter)
Cold Spring Harbor Laboratory
Authorship: John P. Wilson(1)*, Nikita Saha Turna(1), Rosamonde Banks(2), Alexandre Zougman(2), Darryl J.C. Pappin(1)
(1) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; (2) University of Leeds, Leeds, England
| Short Abstract The widespread acceptance and application of proteomics in the clinic requires analytical reproducibility, reliability, low-cost and speed. As molecules with vastly different physiochemical properties, proteins are uniquely challenging. S-Trap sample processing unifies sample solubilization with high concentrations of SDS (5%), affords protein concentration and cleanup inexpensively with standard lab equipment and allows bottom-up proteomics sample prep in around 1 - 2 hrs. Here, we explore S-Trap clinical applications including dried blood spot protein extraction, reproducibility of sample processing and the effect of S-Trap harsh protein denaturation on antibody-antigen interactions, which may alter results in proteomics based clinical assays due to digestion differences. |
Long Abstract
Introduction
Bottom-up proteomics has been long hampered by limitations in sample preparation. The vastly different solubilities of proteins and myriad protocols to dissolve them lead to very different answers depending on what proteins are (or are not) brought into solution. S-Trap sample processing solves this problem by integrating: 1) strong SDS-based protein solubilization (5% SDS) and complete denaturation (pH < 1 and > 70% organic); 2) simultaneous sample concentration and cleanup; and 3) rapid reactor-type protein digestion. The universal S-Trap protocol works without alteration for all kinds of samples and enables reproducible sample processing using standard lab equipment from protein to peptides in around 1 – 2 hrs. It also allows sample preparation in the case of unknown sample composition, for example in submissions to core labs.
In this work, we apply S-Traps to clinical applications. First, we explore the application of S-Traps to dried blood spots examining protein extraction via 5% SDS versus other techniques. Second, we apply S-Trap sample processing to biological fluids and assay the reproducibility and efficiency in a 96-well plate format. Third and finally, we explore the effect of the harsh protein denaturation in S Trap sample processing on antibody-antigen interactions. Especially when endogenous autoantibodies are present, these antibody-antigen interactions have been reported to alter susceptibility to digestion and thus change the results of mass spec based clinical assays.
Methods
Serum and Noviplex plasma prep cards were purchased. S-Traps were purchased from ProtiFi LLC (www.protifi.com) and used per manufacturer's instructions. Blood was spotted per manufacturer's instructions and extracted with 5% SDS or other aqueous buffers. Level of protein extraction and proteins extracted were determined. Serum was processed in a 96-well plate format in replicate and reproducibility was determined. Antibody-antigen pairs were allowed to bind and digested in with or without S-Trap sample processing including harsh denaturation. In all cases, trypsin was used and samples were analyzed by gel and/or Orbitrap MS.
Results
1) Dried blood spots were quickly and most efficiently extracted with 5% SDS. We characterize the differences in extracted proteins.
2) S-Trap sample processing consistently yielded high reproducibility within and between sample sets with average CVs of < 10%.
3) Antibody-antigen interactions were disrupted by the harsh denaturing steps of S-Trap sample processing, facilitating protein digestion and ameliorating artifacts due to antibody binding.
Conclusions & Discussion
In our hands, S-Trap sample processing enables rapid, efficient and reproducible protein extraction, clean up and digestion for bottom-up proteomics analysis of clinical samples. The use of high concentrations of SDS unifies sample solubilization and extraction while simultaneously preventing sample loss during handling. The harsh denaturation conditions equalize digestibility and removes the potential bias of antigens masked by antibody interactions. As a low-cost consumable using only standard lab equipment, we believe this processing strategy will be of use to many.
References & Acknowledgements:
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