Mass spectrometry analysis of soybean seed proteins: optimization of gel-free quantitative workflow

For high-throughput quantitative mass spectrometry (MS) analysis of soybean seed proteins, a method that avoids gel fractionation is advantageous. We developed and optimized a workflow from protein isolation to MS-based quantitation without polyacrylamide matrices. The objective was to quantitatively compare extraction methods to reproducibly arrive at the highest yield and proteome coverage. Beginning with mature soybean seed, we compared four protein extraction methods, employing either TCA/acetone, urea, urea/thiourea, or phenol. Soybean proteins were extracted, quantified for total protein content, and comparatively visualized by Coomassie SDS-PAGE. The phenol extraction method yielded protein concentrations 2 to 7-fold higher than other extraction methods. Comparison of trypsin to protein ratios (1 : 25, 1 : 50, 1 : 75, and 1 : 100) revealed a near linear increase in spectral counts by MS with increasing trypsin levels. In-solution digestion procedures were also compared to determine optimal resuspension and digestion conditions for peptide extraction and quantitation. A resuspension buffer that contained 50 mM Tris-HCl of pH 8.0 and 5 M urea showed the highest spectral counts and protein identifications. The results of this study show the time-honored phenol extraction method consistently and unequivocally yielded the highest amounts of protein from mature soybean seed, and that buffered urea is sufficient for optimal resuspension of precipitated proteins for tryptic digestion and mass spectrometry.