Sample Prep - The Final Frontier in Biomarker Testing

By Nicolai Bache

Biomarker tests have become an important tool in clinical diagnostics. The PSA test evaluates the risk for prostate cancer, and inflammatory and myocardial panels save lives when performed fast enough. Still biomarkers, especially protein biomarkers, are not yet used to their full potential. While novel approaches employ mass spectrometry instead of immunoassay detection, one bottleneck remains: the need for robust, fast, and efficient sample preparation. 

Read more...

Step 1: Improving speed and scale

In a recent publication (1), researchers at the Max-Planck Institute for Biochemistry in Martinsried near Munich presented an optimized sample preparation workflow for biomarker detection by liquid chromatography-mass spectrometry (LC-MS) that demonstrates potential to radically innovate protein based clinical diagnostics. They found that enzymatic digestion time could be reduced from an overnight procedure to just one hour without any impact on biomarker detection. Using the in-Stage Tip (iST) method (2), digestion was performed in a single vial from which peptides could be directly eluted. This way, sample volumes could be cut down to just one microliter. 

Step 2: Removing bias

In standard procedures, depletion and fractionation methods are often used to remove highly abundant proteins and improve sensitivity. The authors found that skipping these methods had little effect on performance, but in contrast revealed proteins that had previously gone undetected, presumably because they had been lost during the procedure. Therefore, they decided to work with the original serum or plasma sample, thereby removing any possible bias

Step 3: Information content vs. time

During the liquid chromatography step, the authors carefully monitored the effect of gradient times on information content. Interestingly, the number of identified proteins decreased only very slowly as a function of shortening the gradient time: Whereas the 100-minute run yielded 1,040 proteins, the 20-minute run already provided information on 450 plasma proteins. More importantly, this subset was sufficient to detect 45 out of 54 FDA-approved “act in plasma” biomarkers, and therefore was chosen as a standard procedure.

Last but not least: Quality Control

The fast, high-throughput procedure allowed to evaluate inter-and intrasample variation and provided information on sample quality that could be related to handling and storage issues, such as hemolysis and partial coagulation in plasma. Potential quality issues were taken into account during data analysis, optimizing biomarker detection even in poor quality samples.

The entire workflow – from sample to data – takes only three hours. This overall increase in speed and robustness is an exciting first step in overcoming the sample prep bottleneck in biomarker detection.

REFERENCES

(1) Geyer, P.E., Kulak, N.A., Pichler, G., Holdt, L.M., Teupser, D., Mann, M. Plasma Proteome Profiling to Assess Human Health and Disease. Cell Systems 2, 185-195, 2016.
http://pubman.mpdl.mpg.de/pubman/item/escidoc:2352959:3/component/escidoc:2352964/1-s2.0-S2405471216300722-main.pdf.

(2) Kulak, N.A., Pichler, G., Paron, I., Nagaraj, N., and Mann, M. (2014) Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.    Nature Methods 11, 319–324.
http://www.nature.com/nmeth/journal/v11/n3/full/nmeth.2834.html

0 Comments

Submit a Comment

Your email address will not be published.

Your comment will be visible after moderation (usually within 24 hours).