Optimization and evaluation of magnetic bead separation combined with matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) for proteins profiling of peritoneal dialysis effluent

Abstract

Peritoneal dialysis effluent (PDE) potentially carries an archive of peptides relevant to pathological processes in abdominal and surrounding tissues. Magnetic beads and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is one such approach that offers a unique tool for profiling of peptides, but this approach has not been used in the PDE analysis. In this study, we developed a strategy for screening PDE proteins <15 kDa and applied this technique to identify potential biomarkers for peritonitis. We examined four kinds of magnetic beads, including a carbon series (C3, C8), weak cation exchange (WCX) and immobilized metal-affinity chromatography (IMAC-Cu) beads. Samples processed with IMAC-Cu magnetic beads consistently showed more MS signals across all beads within the measured mass range. Moreover, there was no difference in the number and morphology of MS signals between concentrated and unconcentrated samples. The PDE peptidome pattern, based on a panel of 15 peaks, accurately recognized peritonitis PD patients from peritonitis-free patients with sensitivity of 90.5% and specificity of 94.7% respectively. Therefore, IMAC-Cu magnetic beads and unconcentrated samples can be used as a fast and cost-effective approach for sample preparation prior to more in-depth discovery of predictive biomarkers of disease in patients on dialysis.

Figure 1.

Protein profiles obtained with various chemical affinity beads. Peptides and proteins were captured by magnetic bead-based technology using C3, C8, weak cation exchange (WCX), and immobilized metal-affinity chromatography (IMAC-Cu) beads. In each panel, the compiled protein spectra from the PD + P and PD − P samples for each bead type are shown with the density plots of individual peritoneal dialysis effluent (PDE) profiles. X-axis, the calculated molecular mass (m/z values); Y-axis, relative intensity of specific samples. The differential density along the x-axis represents the specific peptide that distinctly presents in the samples. IMAC-Cu shows adequate protein profiles across all beads (labeled with a red frame).

Figure 2.

Protein profiles obtained with concentrated and unconcentrated samples. X-axis, the calculated molecular mass (m/z values); Y-axis, relative intensity of specific samples. Based on total signals count, unconcentrated samples consistently demonstrated a similar number of resolved fingerprints within the measured mass range of 1–15 kDa when compared with concentrated samples.

Figure 3.

The intra-day reproducibility study for matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) measurement. The same PDE sample was processed with IMAC-Cu in a single vial and spotted three times on SCOUT 600 μm AnchorChip target positions D1, D2, and D3. Eight MS signals with high, medium, and low abundances within the mass range of m/z 1–15 kDa were randomly selected for the variability assessment. The results are shown in Table 3. ^* selected MS signals.

Figure 4.

The inter-day reproducibility study for MALDI-TOF MS measurement. The sample was split into three vials and processed on different days (marked 101908; 102008; 102108). The variability in signal intensity was estimated across eight MS signals of high, medium, and low abundances within the mass range of m/z 1–15 kDa. The ratios of the signal intensities are listed in Table 4. ^* selected MS signals.

Figure 5.

Discrimination features of peptides with m/z 2230 (x-axis) and 2358 (y-axis) between the patients with peritonitis (red cross) and control group (green circle).