9 research outputs found
Measuring H<sub>2</sub><sup>18</sup>O Tracer Incorporation on a QQQ-MS Platform Provides a Rapid, Transferable Screening Tool for Relative Protein Synthesis
Intracellular proteins are in a state of flux, continually
being
degraded into amino acids and resynthesized into new proteins. The
rate of this biochemical recycling process varies across proteins
and is emerging as an important consideration in drug discovery and
development. Here, we developed a triple-stage quadrupole mass spectrometry
assay based on product ion measurements at unit resolution and H<sub>2</sub><sup>18</sup>O stable tracer incorporation to measure relative
protein synthesis rates. As proof of concept, we selected to measure
the relative in vivo synthesis rate of ApoB100, an apolipoprotein
where elevated levels are associated with an increased risk of coronary
heart disease, in plasma-isolated very low density lipoprotein (VLDL)
and low density lipoprotein (LDL) in a mouse in vivo model. In addition,
serial time points were acquired to measure the relative in vivo synthesis
rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol
transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two
potential therapeutic targets to reduce plasma ApoB100 levels at 2
and 6 h post-tracer-injection. The combination of H<sub>2</sub><sup>18</sup>O tracer with the triple quadrupole mass spectrometry platform
creates an assay that is relatively quick and inexpensive to transfer
across different biological model systems, serving as an ideal rapid
screening tool for relative protein synthesis in response to treatment
Measuring H<sub>2</sub><sup>18</sup>O Tracer Incorporation on a QQQ-MS Platform Provides a Rapid, Transferable Screening Tool for Relative Protein Synthesis
Intracellular proteins are in a state of flux, continually
being
degraded into amino acids and resynthesized into new proteins. The
rate of this biochemical recycling process varies across proteins
and is emerging as an important consideration in drug discovery and
development. Here, we developed a triple-stage quadrupole mass spectrometry
assay based on product ion measurements at unit resolution and H<sub>2</sub><sup>18</sup>O stable tracer incorporation to measure relative
protein synthesis rates. As proof of concept, we selected to measure
the relative in vivo synthesis rate of ApoB100, an apolipoprotein
where elevated levels are associated with an increased risk of coronary
heart disease, in plasma-isolated very low density lipoprotein (VLDL)
and low density lipoprotein (LDL) in a mouse in vivo model. In addition,
serial time points were acquired to measure the relative in vivo synthesis
rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol
transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two
potential therapeutic targets to reduce plasma ApoB100 levels at 2
and 6 h post-tracer-injection. The combination of H<sub>2</sub><sup>18</sup>O tracer with the triple quadrupole mass spectrometry platform
creates an assay that is relatively quick and inexpensive to transfer
across different biological model systems, serving as an ideal rapid
screening tool for relative protein synthesis in response to treatment
High Resolution Discovery Proteomics Reveals Candidate Disease Progression Markers of Alzheimer’s Disease in Human Cerebrospinal Fluid
<div><p>Disease modifying treatments for Alzheimer’s disease (AD) constitute a major goal in medicine. Current trends suggest that biomarkers reflective of AD neuropathology and modifiable by treatment would provide supportive evidence for disease modification. Nevertheless, a lack of quantitative tools to assess disease modifying treatment effects remains a major hurdle. Cerebrospinal fluid (CSF) biochemical markers such as total tau, p-tau and Ab42 are well established markers of AD; however, global quantitative biochemical changes in CSF in AD disease progression remain largely uncharacterized. Here we applied a high resolution open discovery platform, dMS, to profile a cross-sectional cohort of lumbar CSF from post-mortem diagnosed AD patients versus those from non-AD/non-demented (control) patients. Multiple markers were identified to be statistically significant in the cohort tested. We selected two markers SME-1 (p<0.0001) and SME-2 (p = 0.0004) for evaluation in a second independent longitudinal cohort of human CSF from post-mortem diagnosed AD patients and age-matched and case-matched control patients. In cohort-2, SME-1, identified as neuronal secretory protein VGF, and SME-2, identified as neuronal pentraxin receptor-1 (NPTXR), in AD were 21% (p = 0.039) and 17% (p = 0.026) lower, at baseline, respectively, than in controls. Linear mixed model analysis in the longitudinal cohort estimate a decrease in the levels of VGF and NPTXR at the rate of 10.9% and 6.9% per year in the AD patients, whereas both markers increased in controls. Because these markers are detected by mass spectrometry without the need for antibody reagents, targeted MS based assays provide a clear translation path for evaluating selected AD disease-progression markers with high analytical precision in the clinic.</p></div
The demographic characteristics of the longitudinal study population, Cohort-2, at the time of CSF collection.
<p>a: Data at the first visit.</p><p>CTL = control; AD = Alzheimer's disease; N, n = number of subjects/patients; SD = standard deviation; MMSE = Mini- mental state examination; ApoE = Apolipoprotein E</p><p>The demographic characteristics of the longitudinal study population, Cohort-2, at the time of CSF collection.</p
Receiver-operator curves (ROC) for cross-sectional study (Cohort-1).
<p>Comparison of the AD samples versus the control samples was used to estimate the sensitivity, while the comparisons between two groups comprised of randomly selected but equally balanced AD and control samples were used to estimate specificity.</p
The demographic characteristics of the cross sectional study population, Cohort-1, at the time of CSF collection.
<p>CTL = control; AD = Alzheimer's disease; N, n = number of subjects/patients; SD = standard deviation; MMSE = Mini- mental state examination; ApoE = Apolipoprotein E</p><p>The demographic characteristics of the cross sectional study population, Cohort-1, at the time of CSF collection.</p
Quantitation of SME1 and SME2 by dMS (Cohort-1) and 2-way plot.
<p>SME 1 (Peptide NSEPQDEGELFQGVDPR, from neurosecretory protein VGF precursor) and SME2 (peptide VAELEHGSSAYSPPDAFK, from neuronal pentraxin receptor-1) are significantly reduced in AD patients as compared to controls. Area under the curve (AUC) intensity measurements are shown on linear scale. Horizontal bar represents the mean value, vertical error bar represents SD. (A) SME1, P < 0.0001. (B) SME2, P < 0.0005. (C) Two way plot of SME1 and SME2. The symbols (●, +) represent AD and control, respectively.</p
Twenty-five candidate disease progression biomarkers from post-hoc mixed-effect analysis of entire Cohort-2 proteomics data set.
<p>Linear mixed-effect model analysis of log area under the curve (AUC) intensity values for features quantitated by dMS in CSF samples taken annually in longitudinal cohort, Cohort-2. Details described in Methods.</p
Linear mixed effect model analysis of SME1 and SME2 and tau, p-tau, and AB42 in the longitudinal cohort, Cohort-2.
<p><b>SME1:</b> dMS feature ID 751080736 at m/z = 639.63 Da; z = 3; Mo = 1915.85 Da; AA sequence = NSEPQDEGELFQGVDPR; unique to protein sequence IPI00289501.2 Neurosecretory protein VGF.</p><p><b>SME2</b> = dMS feature ID751082515 at m/z = 635.98 Da, z = 3, Mo = 1903.90 Da, AA sequence = VAELEHGSSAYSPPDAFK, unique to protein sequence IPI00334238.1 Neuronal pentraxin receptor 1 (NPTXR).</p><p>Serial CSF samples were taken annually. Log area under the curve (AUC) intensity values for SME1 and SME2 measured by dMS and ELISA measurements for tau, p-tau, and AB42 were analyzed in the mixed-effect model described in methods.</p