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Clinical Trial
. 2015 Aug 13;10(8):e0135365.
doi: 10.1371/journal.pone.0135365. eCollection 2015.

High Resolution Discovery Proteomics Reveals Candidate Disease Progression Markers of Alzheimer's Disease in Human Cerebrospinal Fluid

Ronald C Hendrickson  1 , Anita Y H Lee  1 , Qinghua Song  2 , Andy Liaw  2 , Matt Wiener  3 , Cloud P Paweletz  1 , Jeffrey L Seeburger  4 , Jenny Li  1 , Fanyu Meng  1 , Ekaterina G Deyanova  1 , Matthew T Mazur  1 , Robert E Settlage  1 , Xuemei Zhao  1 , Katie Southwick  1 , Yi Du  1 , Dan Holder  5 , Jeffrey R Sachs  3 , Omar F Laterza  6 , Aimee Dallob  6 , Derek L Chappell  6 , Karen Snyder  6 , Vijay Modur  7 , Elizabeth King  8 , Catharine Joachim  8 , Andrey Y Bondarenko  9 , Mark Shearman  7 , Keith A Soper  5 , A David Smith  8 , William Z Potter  4 , Ken S Koblan  7 , Alan B Sachs  1 , Nathan A Yates  1
Affiliations
Clinical Trial

High Resolution Discovery Proteomics Reveals Candidate Disease Progression Markers of Alzheimer's Disease in Human Cerebrospinal Fluid

Ronald C Hendrickson et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

Competing Interests: All authors performed this study while employees at Merck & Co. or Oxford University or Rosetta Biosoftware. Some authors own stock or stock options in Merck & Co. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Receiver-operator curves (ROC) for cross-sectional study (Cohort-1).
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.
Fig 2
Fig 2. Quantitation of SME1 and SME2 by dMS (Cohort-1) and 2-way plot.
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.
Fig 3
Fig 3. SRM mass spectrometry based assay to quantitate SME1 and SME2 from human CSF.
Example total ion current chromatograms for peptide (A) NSEPQDEGELFQGVDPR from neurosecretory protein VGF (SME1) and (B) the corresponding stable isotope labeled internal standard peptide; and (D) VAELEHGSSAYSPPDAFK from Neuronal Pentraxin Receptor (SME2) and the corresponding internal standard peptide (E) measured by SRM. The observed signal corresponds to the injection of a 4μL equivalent of CSF on column with an internal standard concentration of 26 nM. The peak area (AUCs) measured using LCQuan are shown in grey for each peak. A signal-to-noise ratio measured for the endogenous NSEPQDEGELFQGVDPR and VAELEHGSSAYSPPDAFK was 131 and 63, respectively. Analysis 90 individual technical replicates of 100μL CSF (C) and (F). Response ratios were determined for 90 technical replicates of CSF. Samples were processed in 3 blocks of 30 samples. The mean value (blue line) and standard deviation (red error bars) are shown for each block. The mean value and standard deviation for all samples (black dashed line) is shown on the right hand vertical axis. The coefficient of variation is indicated for each block on the top horizontal axis. Within and between block variability, as determined by ANOVA, is shown on the bottom of each plot.
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Publication types

Grants and funding

This discovery proteomics and analysis work was supported by Merck & Co. The manuscript was reviewed and approved for publication by Merck. The funders had no other role in the study design, data collection and analysis or preparation of the manuscript. The specific roles of the authors are articulated in the "author contribution" section.