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You can’t detect what you don’t inject

by Jessica Nickerson, Research Associate

Precipitation has previously been plagued by low recoveries and biases towards the recovery of hydrophilic, high molecular weight, and acidic proteins. As we know, the mass spectrometry data can only reflect the fraction of the sample that gets injected, so it is critical that every step of the preparative workflow conserves the native profile of the proteome. From extraction to clean-ups to fractionation and enrichment approaches, there are ample opportunities for the proteome profile to become skewed. It is easy to imagine how these types of biases could be extremely problematic in cases where the targeted analyte exhibits relatively low abundance, or where accurate quantitation is needed.

A major goal within the proteomics community is to identify robust diagnostic and prognostic biomarkers, facilitating the detection of hard-to-diagnose diseases. However, a sample as complex as blood, for example, is comprised of 12 proteins that make up 95% of the sample, with the other 5% comprising proteins at concentrations down to 10 orders of magnitude less concentrated than the most abundant ones. Often, the most abundant proteins would be depleted [1], but it is critical that the less abundant proteins are quantitatively conserved across the rest of the workflow in order to optimize the accuracy of the subsequent mass spec characterization.

The ProTrap XG workflow affords the use of efficient extraction buffers, containing up to 1% SDS, with the promise that our optimized precipitation protocol will deplete 99.8% of that SDS, while recovering the proteome quantitatively. The Doucette lab has demonstrated that the inclusion of salt comprehensively recovers a proteome sample with minimal bias towards faster recovery of high molecular weight species, and no statistically significant bias based on hydrophobicity or charge [2,3]. Subsequent work from the group has shown that a step up to 97% acetone and the inclusion of zinc sulfate assures >90% recovery of low molecular weight species down to 2 kDa and near quantitative recovery for peptides as low as 1 kDa [4].

By facilitating an SDS-assisted extraction, there should be no bias towards the recovery of the more hydrophilic fraction of the proteome, and with robust precipitation protocols that recover proteins of all molecular weights down to 1 kDa, a ProTrap XG-based workflow ensures comprehensive recovery of the proteome sample. With quantitative recovery, we optimize the detection efficiency quantitation accuracy of even the least abundant proteins (which are sometimes the most important). Additionally, quantitative recovery helps reduce the limit of detection and quantitation, which, in the case of biomarkers for example, may afford earlier detection of diseases.

[1] Echan, Lynn A, Tang, Hsin-Yao, Ali-Khan, Nadeem, Lee, KiBeom, and Speicher, David W. “Depletion of Multiple High-abundance Proteins Improves Protein Profiling Capacities of Human Serum and Plasma.” Proteomics (Weinheim) 5.13 (2005): 3292-303.
[2] Crowell, Andrew M.J, Wall, Mark J, and Doucette, Alan A. “Maximizing Recovery of Water-soluble Proteins through Acetone Precipitation.” Analytica Chimica Acta 796 (2013): 48-54.
[3] Nickerson, Jessica L, and Doucette, Alan A. “Rapid and Quantitative Protein Precipitation for Proteome Analysis by Mass Spectrometry.” Journal of Proteome Research 19.5 (2020): 2035-042.
[4] Baghalabadi, Venus, and Doucette, Alan A. “Mass Spectrometry Profiling of Low Molecular Weight Proteins and Peptides Isolated by Acetone Precipitation.” Analytica Chimica Acta 1138 (2020): 38-48.