Balancing Deep Proteome Coverage with Limited Sample Amounts Using the Proteograph Product Suite
This application note provides a framework for understanding Proteograph performance when limited starting sample volumes are used.
In this application note, we explore the performance of the Proteograph Product Suite coupled with a state-of-the-art mass spectrometer with conventional nano-flow liquid chromatography workflows (nanoLC-MS/MS) when limited sample volumes are utilized. We evaluated the sample processing of mouse serum starting volumes of 250, 125, 50, 25, and 10 µL, using the SP100 automation instrument. We investigated the LC-MS/MS performance using ~30-minute LC methods coupled to data independent-acquisition strategies (DIA) and analyzed the data via our Proteograph Analysis Suite 2.1 platform, evaluating depth of proteome coverage, dynamic range, peptide yield, and reproducibility of the Proteograph platform.
Introduction
Blood plasma is a rich, readily available biospecimen that is commonly utilized in clinical research. However, plasma proteome research is inherently constrained by the large concentration range of proteins within these samples, which makes detection and quantification of low abundant proteins analytically challenging. The ability to overcome this hurdle while interrogating the plasma proteome deeply and broadly has only been partially addressed by laborious, low throughput and non-scalable workflows. These include techniques such as immune-depletion to remove the topmost abundant proteins from human plasma to facilitate detection of less abundant protein; fractionation schemes to chromatographically separate proteins into less complex fractions; and affinity-based enrichment to selectively capture desired proteins. Seer’s Proteograph Product Suite enables high throughput, in-depth plasma proteome characterization using a panel of proprietary engineered, physicochemically distinct nanoparticles (NPs) to broadly sample and quantify proteins. This panel of NPs provides optimal breadth and depth of protein identifications from plasma or serum, while maintaining precise quantification.
Model organisms, like mice, rats, pigs, and monkeys, are utilized to unveil biological insights to human research. However, model organisms often lack the optimal 250 µL starting plasma volume required to perform the Proteograph Assay. Most animals will go into shock if 25-30% of blood volume is rapidly removed, while almost all will die if more than 40% of blood is rapidly removed. For a typical 25-gram mouse, that would be around 180 – 200 µL of blood. This fact impacts the ability to conduct longitudinal sampling studies for model organisms, for example, collecting limited amounts of blood from the model organism while not sacrificing said model organism.
In this application note, we demonstrate a framework for understanding Proteograph performance when limited starting sample volumes are used, as well as the tradeoffs on assay yield and therefore available injection mass for standard nanoLC-MS/MS workflows.
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