The apolipoprotein E (ApoE) ε4 allele is the strongest risk factor of sporadic Alzheimer's disease (AD), however, the fluid concentrations of ApoE and its different isoforms (ApoE2, ApoE3 and ApoE4) in AD patients and among APOE genotypes (APOE ε2, ε3, ε4) remain controversial. Using a novel mass spectrometry-based method, we quantified total ApoE and specific ApoE isoform concentrations and potential associations with age, cognitive status, cholesterol levels and established AD biomarkers in cerebrospinal fluid (CSF) from AD patients versus non-AD individuals with different APOE genotypes. We also investigated plasma total ApoE and ApoE isoform composition in a subset of these individuals. In total n = 43 AD and n = 43 non-AD subjects were included. We found that CSF and plasma total ApoE levels did not correlate with age or cognitive status and did not differ between AD and non-AD subjects deeming ApoE as an unfit diagnostic marker for AD. Also, whereas CSF ApoE levels did not vary between APOE genotypes APOE ε4 carriers exhibited significantly decreased plasma ApoE levels attributed to a specific decrease in the ApoE4 isoform concentrations. CSF total ApoE concentrations were positively associated with CSF, total tau, tau phosphorylated at Thr181 and Aβ1-42 of which the latter association was weaker and only present in APOE ε4 carriers indicating a differential involvement of ApoE in tau versus Aβ-linked neuropathological processes. Future studies need to elucidate whether the observed plasma ApoE4 deficiency is a life-long condition in APOE ɛ4 carriers and whether this decrease in plasma ApoE predisposes APOE ɛ4 carriers to AD.

Apolipoprotein E (ApoE) is a polymorphic protein that plays a major role in lipid metabolism in the central nervous system and periphery. It has three common allelic isoforms, ApoE2, ApoE3, and ApoE4, that differ in only one or two amino acids. ApoE isoforms have been associated with the occurrence and progression of several pathological conditions, such as coronary atherosclerosis and Alzheimer's disease. The aim of this study was to develop a mass spectrometry (MS)-based assay for absolute quantification of ApoE isoforms in cerebrospinal fluid and plasma samples using isotope-labeled peptides. The assay included five tryptic peptides: CLAVYQAGAR (ApoE2), LGADMEDVCGR (ApoE2 and 3), LAVYQAGAR (ApoE3 and 4), LGADMEDVR (ApoE4), and LGPLVEQGR (total ApoE). Both cerebrospinal fluid and plasma samples were assayed to validate the method. The digestion yield and the extension of chemical modifications in selected amino acid residues (methionine oxidation, glutamine deamidation, and cyclization of N-terminus carbamidomethylcysteine) were also studied. The ApoE phenotype was successfully assigned to all samples analyzed in a blinded manner. The method showed good linearity (R(2) > 0.99) and reproducibility (within laboratory imprecision <13%). The comparison of the MS-based assay with an ELISA for total ApoE concentration showed a moderate correlation (R(2) = 0.59). This MS-based assay can serve as an important tool in clinical studies aiming to elucidate the association between ApoE genotype, total ApoE, and ApoE isoform concentrations in various disorders related to ApoE polymorphisms.

No abstract

No abstract

BACKGROUND:Cancer is a leading cause of death worldwide. The low diagnostic sensitivity and specificity of most current cancer biomarkers make early cancer diagnosis a challenging task. The comprehensive study of peptides and small proteins in a living system, known as "peptidomics," represents an alternative technological approach to the discovery of potential biomarkers for the assessment of a wide variety of pathologies. This review examines the current status of peptidomics for several body fluids, with a focus on urine, for cancer diagnostics applications.CONTENT: Several studies have used high-throughput technologies to characterize the peptide content of different body fluids. Because of its noninvasive collection and high stability, urine is a valuable source of candidate cancer biomarkers. A wide variety of preanalytical issues concerning patient selection and sample handling need to be considered, because not doing so can affect the quality of the results by introducing bias and artifacts. Optimization of both the analytical strategies and the processing of bioinformatics data is also essential to minimize the false-discovery rate.

Background: Alzheimer’s disease (AD) is the most common type of dementia, with progressive onset of clinical symptoms. The main pathological hallmarks are brain deposits of extracellular amyloid beta plaques and intracellular neurofibrillary tangles (NFT). Cerebrospinal fluid reflects pathological changes in the brain; amyloid beta 1-42 is a marker of amyloid plaques, while total and phosphorylated tau are markers of NFT formation. Additional biomarkers associated with disease pathogenesis are needed, for better prognosis, more specific diagnosis, prediction of disease severity and progression and for improved patient classification in clinical trials. The aim of the present study was to evaluate brain-specific proteins as potential biomarkers of progression of AD. Methods: Overall, 30 candidate proteins were quantified in cerebrospinal fluid (CSF) samples from patients with mild cognitive impairment (MCI) and mild, moderate and severe AD dementia (n=101) using mass spectrometry-based selected reaction monitoring assays. ELISA was used for neuronal pentraxin receptor-1 (NPTXR) confirmation. Results: The best discrimination between MCI and more advanced AD stages (moderate and severe dementia) was observed for protein NPTXR (area under the curve, AUC=0.799). A statistically different abundance of this protein was observed between the two groups, with severe AD patients having progressively lower levels (p<0.05). ELISA confirmed lower levels in AD, in a separate cohort that included controls, MCI and AD patients. Conclusions: We conclude that NPTXR protein in CSF is a novel potential biomarker of AD progression and could have important utility in assessing treatment success in clinical trials.

Brain injury is a medical emergency that needs to be diagnosed and treated promptly. Several proteins have been studied as biomarkers of this medical condition. The aims of this study were to: 1) evaluate the selectivity and precision of a commercial ELISA kit for neurofilament medium polypeptide (NFM) protein; and 2) evaluate the concentration in cerebrospinal fluid (CSF) and serum of healthy individuals and patients with brain damage.An ELISA from Elabscience was used. The selectivity was evaluated using size-exclusion chromatography and mass spectrometry. Intra- and inter-batch coefficients of variation (CV) were also studied. Fifty-one CSF samples from 36 age-matched patients with hemorrhagic stroke (HS) (n=30), ischemic stroke (IS) (n=11) and healthy individuals (n=10) were assayed. In addition, serum samples from healthy volunteers (n=47), 68 serum samples from seven patients with HS, 106 serum samples from 12 patients with traumatic brain injury (TBI) and 68 serum samples from 68 patients with mild traumatic brain injury (mTBI) were also analyzed.NFM was identified in the chromatographic fraction with highest immunoreactivity. The intra- and inter-batch CVs were ≤10% and ≤13%, respectively. The CSF-NFM concentration in HS was significantly higher (p<0.0001) than in IS and controls. Serum NFM concentration ranged from 0.26 to 8.57 ng/mL in healthy individuals (median=2.29), from 0.97 to 42.4 ng/mL in HS (median=10.8) and from 3.48 to 45.4 ng/mL in TBI (median=14.7). Finally, 44% of patients with mTBI had increased NFM concentration, with significantly higher levels (p=0.01) in patients with polytrauma.To our knowledge this is the first study describing increased NFM levels in CSF and serum from patients with brain damage.

Cerebrospinal fluid (CSF) is a promising clinical sample for identification of novel biomarkers for various neurological disorders. Considering its direct contact with brain tissue, CSF represents a valuable source of brain-related and brain-specific proteins. Multiple sclerosis is an inflammatory, demyelinating neurological disease affecting the central nervous system, and so far there are no diagnostic or prognostic disease specific biomarkers available in the clinic. The primary aim of the present study was to develop a targeted mass spectrometry assay for simultaneous quantification of 30 brain-related proteins in CSF and subsequently to demonstrate assay feasibility in neurological samples derived from multiple sclerosis patients. Our multiplex selected reaction monitoring assay had wide dynamic range (median fold range across peptides = 8.16 × 10) and high assay reproducibility (median across peptides CV = 4%). Candidate biomarkers were quantified in CSF samples from neurologically healthy individuals (n = 9) and patients diagnosed with clinically isolated syndrome (n = 29) or early multiple sclerosis (n = 15).