Figure 8.

Scheme showing the process of analyzing a human serum sample by mass spectrometry. The serum sample is diluted with the chaotrope guanidinium chloride (GuCl; 6 M) and the sample is then reduced/alkylated with dithiothreitol (DTT) and iodoacetamide (IA), respectively. The sample is methanol precipitated and then brought up in 90 microliters of buffer (8% acetonitrile, 0.01% BOG, 25 mM ABC pH 8) and then digested with trypsin. The trypsin digest is filtered sequentially through a 10,000 and a 5000 Da MWCO filter. The internal standard (IS) is added and the sample is run on the instrument. The appropriate ¹⁵N-labeled internal standard is used to identify the peak corresponding to analogous unlabeled peptide based on their identical physico-chemical properties (chromatographic retention time and MS fragmentation). The area ratio of the unlabeled peptide to that of the added internal standard may be calculated and used to quantitate the amount of Stx present in the sample. The signal shown is from a human serum sample spiked with Stx2g.