13 research outputs found
Quantification of Botulinum Neurotoxin Serotypes A and B from Serum Using Mass Spectrometry
Botulinum neurotoxins (BoNT) are the deadliest agents known. Previously, we reported an endopeptidase activity based method (Endopep-MS) that detects and differentiates BoNT serotypes A–G. This method uses serotype specific monoclonal antibodies and the specific enzymatic activity of BoNT against peptide substrates which mimic the toxin’s natural target. Cleavage products from the reaction are detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We have now developed a multiple reaction monitoring method to quantify the biological activity of BoNT serotypes A (BoNT/A) and B (BoNT/B) present in 0.5 mL of serum using electrospray mass spectrometry. The limit of quantification for each serotype is 1 mouse intraperitoneal lethal dose (MIPLD<sub>50</sub>) corresponding to 31 pg of BoNT/A and 15 pg of BoNT/B in this study. This method was applied to serum from rhesus macaques with inhalational botulism following exposure to BoNT/B, showing a maximum activity of 6.0 MIPLD<sub>50</sub>/mL in surviving animals and 653.6 MIPLD<sub>50</sub>/mL in animals that died in the study. The method detects BoNT/B in serum 2–5 h after exposure and up to 14 days. This is the first report of a quantitative method with sufficient sensitivity, selectivity, and low sample size requirements to measure circulating BoNT activity at multiple times during the course of botulism
Core lipid, surface lipid and apolipoprotein composition analysis of lipoprotein particles as a function of particle size in one workflow integrating asymmetric flow field-flow fractionation and liquid chromatography-tandem mass spectrometry
<div><p>Lipoproteins are complex molecular assemblies that are key participants in the intricate cascade of extracellular lipid metabolism with important consequences in the formation of atherosclerotic lesions and the development of cardiovascular disease. Multiplexed mass spectrometry (MS) techniques have substantially improved the ability to characterize the composition of lipoproteins. However, these advanced MS techniques are limited by traditional pre-analytical fractionation techniques that compromise the structural integrity of lipoprotein particles during separation from serum or plasma. In this work, we applied a highly effective and gentle hydrodynamic size based fractionation technique, asymmetric flow field-flow fractionation (AF4), and integrated it into a comprehensive tandem mass spectrometry based workflow that was used for the measurement of apolipoproteins (apos A-I, A-II, A-IV, B, C-I, C-II, C-III and E), free cholesterol (FC), cholesterol esters (CE), triglycerides (TG), and phospholipids (PL) (phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC)). Hydrodynamic size in each of 40 size fractions separated by AF4 was measured by dynamic light scattering. Measuring all major lipids and apolipoproteins in each size fraction and in the whole serum, using total of 0.1 ml, allowed the volumetric calculation of lipoprotein particle numbers and expression of composition in molar analyte per particle number ratios. Measurements in 110 serum samples showed substantive differences between size fractions of HDL and LDL. Lipoprotein composition within size fractions was expressed in molar ratios of analytes (A-I/A-II, C-II/C-I, C-II/C-III. E/C-III, FC/PL, SM/PL, PE/PL, and PI/PL), showing differences in sample categories with combinations of normal and high levels of Total-C and/or Total-TG. The agreement with previous studies indirectly validates the AF4-LC-MS/MS approach and demonstrates the potential of this workflow for characterization of lipoprotein composition in clinical studies using small volumes of archived frozen samples.</p></div
Average lipid/Lp-P vs. size profiles.
<p>A: HDL particle size range, B: LDL particle size range. Error bars indicate 95% confidence intervals based on 110 serum samples. Vertical dashed lines indicated the borders of size ranges with significantly different composition as discussed in the text. Corresponding size ranges are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194797#pone.0194797.g009" target="_blank">Fig 9</a>.</p
Sum of ApoA-I, sum of calculated Lp-P, average ApoA-I/Lp-P and other average HDL particle characteristics by diameter range in different sample categories.
<p>Labels indicate normolipidemic (NL, N = 25, Total-C <230 mg/dL, and Total-TG <150 mg/dL); hypercholesterolemic (HC, N = 13, Total-C >230 mg/dL and Total-TG <150 mg/dL); hyperlipidemic (HL, N = 41, Total-C >230 mg/dL and Total-TG >150 mg/dL); and hypertriglyceridemic (HT, N = 31, Total-C<230 mg/dL and Total-TG>150 mg/dL). Error bars indicate standard deviation.</p
Average number of apo/Lp-P vs. size profiles.
<p>Error bars indicate 95% confidence intervals based on 110 serum samples. Vertical dashed lines indicated the borders of size ranges with significantly different composition as discussed in the text. Corresponding size ranges are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194797#pone.0194797.g010" target="_blank">Fig 10</a>.</p
Average exchangeable apo/apoB-100 molar ratio profiles, and protein volume vs. total particle volume ratios by sample categories.
<p>Labels indicate normolipidemic (NL, N = 25, Total-C <230 mg/dL, and Total-TG <150 mg/dL); hypercholesterolemic (HC, N = 13, Total-C >230 mg/dL and Total-TG <150 mg/dL); hyperlipidemic (HL, N = 41, Total-C >230 mg/dL and Total-TG >150 mg/dL); and hypertriglyceridemic (HT, N = 31, Total-C<230 mg/dL and Total-TG>150 mg/dL). Error bars indicate confidence intervals.</p
Overlay of size distribution profiles of selected proteins from a single sample.
<p>LC-MS/MS peak areas were normalized by the maximum peak area for overlay.</p
Size profiles from serum and corresponding fractions.
<p><b>A</b>: Size profile of representative proteins from serum (50 μL injection volume). <b>B</b>: Size profile from re-injected AF4 fractions indicated by colors (200 μL injection volume). <b>C</b>: De-convolution of the apoA-I profile in A (bottom) and predicted fraction profiles (top). <b>D</b>: De-convolution of the apoB profile in A (bottom) and predicted fraction profiles (top), generated by summing together de-convoluted Gaussian peaks. Re-injected fractions are indicated by numbers on the top and by the colors of solid vertical lines in <b>A</b>, matched with the color of the overlaid size profiles in <b>B</b>. The dotted vertical lines in <b>B</b> correspond with lines in <b>A</b>. The colors of the predicted profiles in <b>C</b> and <b>D</b> matched with the colors of the size profiles of the fractions for apoA-I and apoB in <b>B</b>.</p
Overlay of size distribution profiles of apolipoproteins and lipids for representative samples from different categories.
<p>Labels indicate normolipidemic (NL, Total-C = 206 mg/dL and Total-TG = 130 mg/dL), hypercholesterolemic (HC, Total-C = 249 mg/dL and Total-TG = 121 mg/dL); hyperlipidemic (HL, Total-C = 287 mg/dL and Total-TG = 180 mg/dL); and hypertriglyceridemic (HT, Total-C = 212 mg/dL and Total-TG = 188 mg/dL). Y axis for apolipoprotein concentrations are in nM and for lipids are in μM units.</p
Summary of mean and standard deviation of analyte levels in whole serum for non-polar lipids, polar lipids and proteins by sample categories.
<p>Summary of mean and standard deviation of analyte levels in whole serum for non-polar lipids, polar lipids and proteins by sample categories.</p