Although blood is a good starting material for biomarker discovery, high-abundance proteins (HAPs) such as albumin threaten to mask changes in LMW compounds below 30 kDa. The most valuable biomarkers are often low-abundance LMW proteins, which show variation in terms of quantity, post-translational modification and isoform expression according to disease. Quantitative proteomic analysis using liquid chromatography–mass spectrometry (LC-MS) characterization is thus a valuable tool in biomarker discovery, since it is sensitive enough for both detection and quantitation. However, the necessary removal of HAPs during sample preparation often results in loss of LMW proteins in addition to incomplete removal, masking their quantitative evaluation.
Chen et al. propose a gel filtration enrichment step during sample preparation that passively sieves out the HAPs using a four-layer tracking tricine sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) technique. The technique uses the following four layers:
- A standard stacking layer (5%)
- A funnel layer (8%–10%) that prevents more abundant proteins from clogging up the interface
- A blocking layer (16%–20%) that retains the high–molecular weight proteins
- A separating gel (10%–12%) that presents the LMW proteins as separate bands for in-gel trypsin digestion
The researchers obtained serum samples from six healthy volunteers (three male, three female), pooling the volumes to minimize variation. They then took aliquots of the pooled sample and prepared them according to the following workflows:
- Denaturing glycine SDS-PAGE
- Optimized differential solubilization (ODS) and PAGE
- Gel filtration enrichment: four-layer stacking tricine SDS-PAGE
Once the researchers separated the proteins, they digested the proteins using trypsin prior to LC–tandem mass spectrometry evaluation (LC-MS/MS) using an LTQ Obitrap Velos mass spectrometer (Thermo Scientific).
The team also used a commercial bead enrichment kit to separate proteins prior to trypsin digestion and LC-MS/MS. They conducted recovery assessment for this method in addition to gel filtration enrichment and ODS using a stable isotope labeling in culture absolute quantitation (SILAC-AQUA) approach to label recombinant proteins, which they then spiked into the human serum before processing according to the workflows described above.
On direct examination of the gels, Chen et al. found that the gel filtration enrichment showed better resolution of LMWs than ODS or glycine SDS-PAGE, with no loss of protein bands. The team found that the gel filtration enrichment resulted in a clear demarcation between blocking and separating gel interfaces. Furthermore, band density varied according to sample load, although volumes above 10 µL resulted in saturation with merging into the separation layer. Compared to the glycine and ODS methods, gel filtration enrichment was more consistent and highly efficient, resulting in more proteins, as shown by band intensity.
The team confirmed this using LC-MS/MS, where final analysis yielded more protein identifications from the gel filtration enrichment (1,576) than from the other two workflows (ODS = 1,022; glycine = 1,186) in 10 µL of serum. Of the 1,576 proteins identified, 559 were LMW below 30 kDa. Gel filtration enrichment also gave good recoveries from spiked samples as assessed by SILAC-AQUA. Compared to ODS (18.7%) and the commercial kit (9.6%), gel filtration enrichment resulted in recoveries of 33.1%.
Chen et al. conclude that quantitative LC-MS/MS shows that gel filtration results in good enrichment without protein bias. Furthermore, the workflow efficiently captures the LMW proteins of interest for biomarker discovery without excessive losses during preparation. Removal of HAPs removes interference with quantitative proteomic evaluation, thus increasing the value of this method for biomarker characterization.
Reference
1. Chen L., et al. (2015) “Development of gel-filter method for high enrichment of low-molecular weight proteins from serum,” PLoS ONE 10(2): e0115862. doi: 10.1371/journal.pone.0115862
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