Figure 1.
Proteins retained in the SAX resin are extracted by stepwise elution. Two aliquots of these extracts are digested with two different enzymes (trypsin and GluC, respectively) and peptides identified by MDLC-ESIMS/MS. Target peptides were confirmed by selected reaction monitoring. The identification of some proteins as removed from patients' blood (and not from the commercial albumin solution used in the MARS circuit) was further validated by Western blot.
Table 1.
Data set of proteins captured by MARS.
Table 2.
Peptides and proteins monitored by SRM (Full data in Table S3).
Figure 2.
Ion chromatograms for several peptide targets in control human serum albumin (HSA) and patient-derived (MARS) extracts (x-axis range of 8 min).
The following SRM transitions were monitored: 1217.5→1298.2, 1441.8 (SLURP1), 1304.4→1739.6, 1080.5, (HNP-1), 1236.6→1183.5, 1515.5 (A1BG) and 1192.9→1969.5, 1683.5 (APOA2) (dotted and solid lines, respectively).
Figure 3.
Confirmation of SLURP1 and HNP-1 by Western blot.
Lanes 1, 2, 4 and 5 are patients with resistant pruritus; Lane 3 is a patient with Wilson's disease. Lane 6 corresponds to the untreated albumin-derived extracts.
Figure 4.
Detection of SLURP1 in serum by Western blot.
Lane 1, positive control; Lanes 2 and 4, serum from healthy people; Lanes 3 and 5, patients with cholestasis and pruritus. The graph represents the normalized Western blot intensities of SLURP1 in four groups: healthy people, patients with cholestasis and pruritus (Chol_P), patients with cholestasis without pruritus (Chol_NP), and patients with cholestasis and pruritus who were taking rifampicin (Chol_P+Rif).
Figure 5.
Mass and location distribution of proteins unique to MARS extracts after patient treatment, in comparison with proteins in commercial albumin and in the human plasma database.