A Novel Peptidome Technology for the Diagnosis of Mild Cognitive Impairment and Alzheimer's Disease by Selected Reaction Monitoring.

Background: With the aging of populations worldwide, Alzheimer's disease (AD) has become a concern due to its high prevalence and the continued lack of established treatments. Early diagnosis is required as a preventive intervention to modify the disease's progression. In our previous study, we performed peptidomic analysis of serum samples obtained from AD patients and age-matched healthy subjects to seek peptide biomarker candidates for AD by using BLOTCHIP-MS analysis, and identified four peptides as AD biomarker candidates. Objective: The objective was to validate the serum biomarker peptides to distinguish mild cognitive impairment (MCI) and AD in comparison to cognitively healthy controls using a new peptidome technology, the Dementia Risk Test. Methods: We enrolled 195 subjects with normal cognitive function (NC; n = 70), MCI (n = 55), and AD (n = 70), The concentrations of cognitive impairment marker peptides (Fibrinogen α chain (FAC), Fibrinogen ß chain (FBC), Plasma protease C1 inhibitor (PPC1I), α2-HS-glycoprotein (AHSG)) were quantified by using a selected reaction monitoring assay based on liquid chromatography-MS/MS. Results: The present study confirmed that three peptides, FAC, FBC, and PPC1I, were significantly upregulated during the onset of AD. This three-peptide set was both highly sensitive in determining AD (sensitivity: 85.7%, specificity: 95.7%, AUC: 0.900) and useful in distinguishing MCI (sensitivity: 61.8%, specificity: 98.6%, AUC: 0.824) from NC. Conclusions: In this validation study, we confirmed the high diagnostic potential of the three peptides identified in our previous study as candidate serum biomarkers for AD. The Dementia Risk Test may be a powerful tool for detecting AD-related pathological changes.

A New Serum Biomarker Set to Detect Mild Cognitive Impairment and Alzheimer's Disease by Peptidome Technology.

BACKGROUND: Because dementia is an emerging problem in the world, biochemical markers of cerebrospinal fluid (CSF) and radio-isotopic analyses are helpful for diagnosing Alzheimer's disease (AD). Although blood sample is more feasible and plausible than CSF or radiological biomarkers for screening potential AD, measurements of serum amyloid- ß (Aß), plasma tau, and serum antibodies for Aß1 - 42 are not yet well established. OBJECTIVE: We aimed to identify a new serum biomarker to detect mild cognitive impairment (MCI) and AD in comparison to cognitively healthy control by a new peptidome technology. METHODS: With only 1.5µl of serum, we examined a new target plate "BLOTCHIP®" plus a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) to discriminate control (n = 100), MCI (n = 60), and AD (n = 99). In some subjects, cognitive Mini-Mental State Examination (MMSE) were compared to positron emission tomography (PET) with Pittsburgh compound B (PiB) and the serum probability of dementia (SPD). The mother proteins of candidate serum peptides were examined in autopsied AD brains. RESULTS: Apart from Aß or tau, the present study discovered a new diagnostic 4-peptides-set biomarker for discriminating control, MCI, and AD with 87% of sensitivity and 65% of specificity between control and AD (***p < 0.001). MMSE score was well correlated to brain Aß deposition and to SPD of AD. The mother proteins of the four peptides were upregulated for coagulation, complement, and plasticity (three proteins), and was downregulated for anti-inflammation (one protein) in AD brains. CONCLUSION: The present serum biomarker set provides a new, rapid, non-invasive, highly quantitative and low-cost clinical application for dementia screening, and also suggests an alternative pathomechanism of AD for neuroinflammation and neurovascular unit damage.

Selected reaction monitoring for colorectal cancer diagnosis using a set of five serum peptides identified by BLOTCHIP®-MS analysis.

BACKGROUND: Colorectal cancer (CRC) is one of the most predominant types of cancer, and it is the fourth most common cause of cancer-related death and it is important to diagnose CRC in early stage to decrease the mortality by CRC. In our previous study, we identified a combination of five peptides as a biomarker candidate to diagnose CRC by BLOTCHIP®-MS analysis using a set of healthy control subjects and CRC patients (stage II-IV). The aim of the present study was to validate the serum biomarker peptides reported in our previous study using a second cohort and to establish their potential usefulness in CRC diagnosis. METHODS: A total of 56 patients with CRC (n = 14 each of stages I-IV), 60 healthy controls, and 60 patients with colonic adenoma were included in this study. The five peptides were extracted and analyzed by selected reaction monitoring using ProtoKey® Colorectal Cancer Risk Test Kit (Protosera, Inc., Amagasaki, Japan). RESULTS: The results clearly showed that the four CRC groups, stages I-IV, could be sufficiently discriminated from the control group and colonic polyp group. This five-peptide set could identify CRC at each stage compared to the control population in this validation cohort, including those with early-stage disease. The AUC values for each stage of CRC compared to the control population were 0.779, 0.946, 0.852, and 0.973 for stages I, II, III, and IV, respectively. CONCLUSIONS: In this case-control validation study, we confirmed high diagnostic performance for CRC using five peptides that were identified in our previous study as serum biomarker candidates for the detection of CRC.

Quantitative peptidomic analysis by a newly developed one-step direct transfer technology without depletion of major blood proteins: its potential utility for monitoring of pathophysiological status in pregnancy-induced hypertension.

We have recently developed a new target plate (BLOTCHIP®) for MALDI-MS. An advantage of this procedure is that it does not require the lowering of protein concentrations in test samples prior to analysis. Accordingly, this new technology enables the detection of peptides present in blood samples, including those that would otherwise be adsorbed to abundant blood proteins and would thus escape detection. Using this technology, we analyzed the peripheral blood of patients with pregnancy-induced hypertension (PIH; the most common serious complication of pregnancy) to test a potential utility of the technology for monitoring of the pathophysiological status. In the present study, we found 23 characteristic peptides for PIH in the blood serum of pregnant women. Offline LC-MALDI MS/MS identified 7 of the 23 peptides as fragments derived from kininogen-1 (three peptides), fibrinogen-α, complement component C4-A/B, α-2-HS-glycoprotein and inter-α-trypsin inhibitor heavy chain H4. 2-D scatter plots with combinations of the peptides found in the present study can be grouped for pregnant women with/without PIH, which would be satisfactory reflected for their status. Additionally, the levels of most of these peptides found were significantly decreased by albumin/IgG depletion prior to BLOTCHIP® analysis in accordance with conventional proteomics procedures. These results indicated that BLOTCHIP® analysis can be applied for discovery study of PIH biomarker candidates.