Figure 1 Total ion chromatogram of crude serum organic extract. (A) Total ion current of bulk serum following liquid/liquid extraction and HPLC-coupled mass spectrometry as explained in the methods. (B) Extracted mass spectra of all masses from (A). (C) Extracted ion chromatograms of GTAs 446, 448 and 450 from the total ion current shown in A. (D) Cell proliferation, as assayed by MTT, for SW620 cells treated with up to 80 ug/ml of the crude serum extract. Organic serum extract was next subjected to flash
column chromatography as described in the methods, resulting in 12 this website fractions which were subsequently analyzed by MEK activation HPLC-MS to determine GTA content. Although other components were present in all the fractions, only fraction 9 out of the 12 was enriched for the C28 GTAs (referred to as the GTA+ve fraction). A GTA negative control fraction (fraction 8, lacking any detectable GTAs) was also selected ICG-001 for the studies described below. Representative total ion chromatograms, extracted mass spectra and selected ion chromatograms of the three C28 GTAs for the GTA-ve and GTA+ve fractions are shown in Figures 2A and 2B, respectively. By comparing the sums of the selected ion chromatograms of the three GTAs to the total ion currents, we estimated that the GTA+ve fraction contained approximately 21% C28 GTAs while the GTA-ve fraction had no detectable
levels (bottom panel of Figures 2A and 2B). The non-GTA background components for both fractions were similar, and the most abundant non-GTA components in the GTA+ve fraction were also the most abundant components in the GTA-ve fraction. Therefore, the two fractions were compositionally similar
other than the 21% GTA content of the GTA+ve fraction, which represented an approximately 143-fold enrichment Non-specific serine/threonine protein kinase of the three C28 GTA metabolites over the crude organic serum extract (as shown in Figure 1A). These fractionations were repeated several times with consistent results. We therefore concluded that the fractions were sufficiently matched for investigating biological activity as described below. For comparison, the relative levels of the three C28 GTAs from 40 pooled CRC patients’ serum and serum from 40 matched control subjects is shown in Figure 2C. Figure 2 Mass spectrometry characterization of semi-purified GTA-ve and GTA+ve extracts. (A) Crude serum extract (as shown in Figure 1) was subject to flash column chromatography as described in the methods resulting in two adjacent eluates, one positive and one negative for the presence of GTAs. The total ion chromatogram (top), extracted mass spectra (middle), and extracted ion chromatograms for three GTAs (GTA446, 448 and 450; bottom) of the GTA-ve fraction. (B) Same as (A) for the GTA+ve fraction. (C) For comparison, the extracted ion chromatograms of GTA446, 448 and 450 from the extracts of serum pooled from 20 CRC patients and 20 controls is shown.