Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of proteins and peptides was performed on samples deposited onto non-porous ether-type polyurethane (PU) membranes. Spectra obtained using PU membranes showed that mass resolution and accuracy were equivalent to values observed using a metal target, and superior to those obtained using poly(vinylidene difluoride) (PVDF) membranes. A small apparent increase in the mass of proteins and also loss of resolution were observed at very high laser irradiance due to charging, but were not observed under normal conditions. Analysis of NaCl-doped standards demonstrated that PU membranes yielded better results than a metallic target for salt-containing solutions. Relatively strong hydrophobic interactions between the proteins and peptides and the PU membrane allowed the incorporation of a washing step. This step allowed for the removal of salts and buffer components and thus provided an increase in resolution and mass accuracy. Digestion of citrate synthase (a protein of molecular weight 47 886) with trypsin was performed directly on the surface of the membrane for variable periods of time, and characteristic peptide fragments were observed by MALDI-TOFMS. Delayed extraction was used to increase the resolution and to permit more accurate mass assignments for those fragments. The use of PU membranes for MALDI-TOFMS analysis of proteins with higher molecular weights is also demonstrated.
A new method for the sampling and off-site analysis of hemoglobin variants by mass spectrometry is reported. This technique uses a nonporous polyurethane membrane as the collection device and transportation medium of a blood sample for analysis. The same membrane is then used as the MALDI-TOF MS sample support for mass spectrometric analysis. Minimal invasive sample collection is afforded by collecting less than 1 µL of blood using a common lancet device. MALDI-TOF MS is performed directly on the membrane, after washing off the interfering plasma components, followed by the addition of matrix. This reduces the time of analysis and prevents sample loss. Enzymatic digestion can be performed directly on the membrane, using in this case trypsin, allowing for further characterization of the sample. The method is much less invasive compared to drawing blood with a syringe. The sample may be transported to the laboratory by regular mail, and thus the method can serve remote locations. We demonstrate the procedure by characterizing the Hb Shepherds Bush hemoglobin variant, b74-(E18)GlyfAsp.
Plasma high-density lipoprotein is commonly estimated by measuring the cholesterol remaining in plasma supernatant solutions after other lipoproteins, which contain apolipoprotein B, are precipitated with heparin and Mn2+. The method (method I) now in use by the Lipid Research Clinics, in which Mn2+ is at 46 mmol/liter final concentration, is reasonably accurate, but precipitation and sedimentation of lipoproteins other than high-density lipoproteins is often incomplete. We evaluated two modifications of method I. In method II, the Mn2+ concentration was doubled; the second modification (method III) included the increased Mn2+ concentration in a combined heparin Mn2+ reagent, decreased sample volume (2 ml), and a shorter incubation time (10 min at room temperature). The percentages of samples with turbid supernates (i.e., incomplete sedimentation) by methods I, II, and III were 9, 3, and 2%, respectively. Among non-turbid supernates, the percentages of samples containing measurable apolipoprotein B (incomplete precipitation) were 79, 19, and 16%, respectively. We conclude that method III is the most convenient and accurate of the three procedures.
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