Apolipoprotein A-I: Insights from redox proteomics for its role in neurodegeneration

Proteomics has a wide range of applications, including determination of differences in the proteome in terms of expression and post-translational protein modifications. Redox proteomics allows the identification of specific targets of protein oxidation in a biological sample. Using proteomic techniques, apolipoprotein A-I (ApoA-I) has been found at decreased levels in subjects with a variety of neurodegenerative disorders including in the serum and cerebrospinal fluid (CSF) of Alzheimer disease (AD), Parkinson disease (PD), and Down syndrome (DS) with gout subjects. ApoA-I plays roles in cholesterol transport and regulation of inflammation. Redox proteomics further showed ApoA-I to be highly oxidatively modified and particularly susceptible to modification by 4-hydroxy-2-trans-nonenal (HNE), a lipid peroxidation product. In the current review, we discuss the consequences of oxidation of ApoA-I in terms of neurodegeneration. ROS-associated chemotherapy related ApoA-I oxidation leads to elevation of peripheral levels of tumor necrosis factor-α (TNF-α) that can cross the blood-brain barrier (BBB) causing a signaling cascade that can contribute to neuronal death, likely a contributor to what patients refer to as “chemobrain.” Current evidence suggests ApoA-I to be a promising diagnostic marker as well as a potential target for therapeutic strategies in these neurodegenerative disorders.     

Identification and validation of novel CSF biomarkers for early stages of Alzheimer's disease

The pathology of Alzheimer's disease (AD) begins years prior to clinical diagnosis. The development of antecedent biomarkers that indicate the presence of AD pathology and predict risk for decline in both cognitively normal and mildly impaired individuals will be useful as effective therapies are developed. While cerebrospinal fluid (CSF) markers such as amyloid-β (Aβ) 42 and tau are useful, additional biomarkers are needed. To identify new markers, we utilized 2-D difference gel electrophoresis (2-D DIGE) of individual CSF samples from subjects with very mild AD versus controls after depletion of high-abundant proteins. Protein spots displaying differential abundance between the two groups were identified with MS. A number of candidate biomarkers were identified in 18 gel features. Selected candidates were quantified in a larger clinical set using ELISA. The mean levels of α1-antichymotrypsin (ACT), antithrombin III (ATIII), and zinc-α2-glycoprotein (ZAG) were significantly higher in the mild AD group, and the mean level of carnosinase 1 (CNDP1) was decreased. When these biomarkers are optimally combined, there is a strong trend toward greater specificity and sensitivity based on clinical diagnosis than when used individually. Our findings provide novel biomarker candidates for very mild and mild AD that can be further assessed as antecedent markers and predictors of clinical progression.     

Mass spectrometry-based proteomics of endoscopically collected pancreatic fluid in chronic pancreatitis research

MS-based investigation of pancreatic fluid enables the high-throughput identification of proteins present in the pancreatic secretome. Pancreatic fluid is a complex admixture of digestive, inflammatory, and other proteins secreted by the pancreas into the duodenum, and thus is amenable to MS-based proteomic analysis. Recent advances in endoscopic techniques, in particular the endoscopic pancreatic function test (ePFT), have improved the collection methodology of pancreatic fluid for proteomic analysis. Here, we provide an overview of MS-based proteomic techniques as applied to the study of pancreatic fluid. We address sample collection, protein extraction, MS sample preparation and analysis, and bioinformatic approaches, and summarize current MS-based investigations of pancreatic fluid. We then examine the limitations and the future potential of such technologies in the investigation of pancreatic disease. We conclude that pancreatic fluid represents a rich reservoir of potential biomarkers and that the study of the molecular mechanisms of chronic pancreatitis may benefit substantially from MS-based proteomics.     

Mass Spectrometric Analysis of Cerebrospinal Fluid Ubiquitin in Alzheimer's Disease and Parkinsonian Disorders

1 Purpose

Dysfunctional proteostasis, with decreased protein degradation and an accumulation of ubiquitin into aggregated protein inclusions, is a feature of neurodegenerative diseases. Identifying new potential biomarkers in cerebrospinal fluid (CSF) reflecting this process could contribute important information on pathophysiology.

2 Experimental design

A developed method combining SPE and PRM‐MS is employed to monitor the concentration of ubiquitin in CSF from subjects with Alzheimer's disease (AD), Parkinson's disease (PD), and progressive supranuclear palsy (PSP). Four independent cross‐sectional studies are conducted, studies 1–4, including controls (n = 86) and participants with AD (n = 60), PD (n = 15), and PSP (n = 11).

3 Results

The method shows a repeatability and intermediate precision not exceeding 6.1 and 7.9%, respectively. The determined LOD is 0.1 nm and the LOQ range between 0.625 and 80 nm . The CSF ubiquitin concentration is 1.2–1.5‐fold higher in AD patients compared with controls in the three independent AD‐control studies (Study 1, p < 0.001; Study 2, p < 0.001; and Study 3, p = 0.003). In the fourth study, there is no difference in PD or PSP, compared to controls.

4 Conclusion and clinical relevance

CSF ubiquitin may reflect dysfunctional proteostasis in AD. The described method can be used for further exploration of ubiquitin as a potential biomarker in neurodegenerative diseases.     

Fully Automated Segmentation of Cerebral Ventricles from 3-D SPGR MR Images using Fuzzy Representative Line

Generating surface shaded display images and measuring the volumes of cerebral ventricles using 3-D SPGR MR images will help to diagnose many types of cerebral diseases with quantitatively and qualitatively. However, manual segmentation of cerebral ventricles is time-consuming and is subject to inter- and intra-operator variation. This article proposes a fully automated method for segmenting cerebrospinal fluid (CSF) and cerebral ventricles from MR images. Our method segments the cerebral ventricles by using a representative line (RL), which can represent the abstract of the shape and position of the cerebral ventricles. The RL is found by fuzzy If-Then rules that can implement physicians’ knowledge on the cerebral ventricles. The proposed method was applied to MR volumes of 20 normal subjects, 20 Alzheimer disease (AD) and 20 normal pressure hydrocephalus (NPH) patients. The segmentation error ratio of the lateral ventricles was 1.98% in comparison with the volumes of manually delineated region by a physician. Using the proposed method, we found that patients of NPH significantly increased the ratio of volume of the lateral ventricles to the total CSF volume in comparison with that of AD (significance level < 0.001)     

Elevated haptoglobin level of cerebrospinal fluid in Guillain-Barré syndrome revealed by proteomics analysis

Guillain‐Barré Syndrome (GBS) is a rare autoimmune inflammatory polyneuropathy with a high risk of respiratory failure and unclear pathogenesis. Currently, there are no valid biomarkers for diagnosis of GBS. We used 2‐DE and MS to analyze the protein profiles of five pairs of cerebrospinal fluid (CSF) samples of the GBS patients and the patient controls. Three proteins (orosomucoid, haptoglobin and apolipoprotein A‐IV) were up‐regulated, and two proteins (prostaglandin D2 synthase and transthyretin) were down‐regulated in the CSF of the GBS patients. The CSF haptoglobin level, quantified by enzyme‐linked immunosorbent assay, was significantly higher in the GBS patients (12.44 ± 2.70 μg/mL) compared to the chronic inflammatory demyelinating polyradiculoneuropathy (2.82 ± 0.83 μg/mL), viral meningitis (3.57 ± 0.97 μg/mL) and control patients (1.44 ± 0.35 μg/mL, p<0.05). This study indicated that protein profile analysis using a combination of 2‐DE and MS provides an effective strategy for elucidating the pathogenesis and identifying potential CSF biomarkers for GBS. The raised intrathecal synthesis of haptoglobin specifically only in GBS patients, but not in patients with other neurological diseases examined, provides evidence of central nervous system involvement in GBS, and may be used as a potential diagnostic marker for GBS.     

Proteomic and biomarker studies and neurological complications of pediatric sickle cell disease

Biomarker analysis and proteomic discovery in pediatric sickle cell disease has the potential to lead to important discoveries and improve care. The aim of this review article is to describe proteomic and biomarker articles involving neurological and developmental complications in this population. A systematic review was conducted to identify relevant research publications. Articles were selected for children under the age of 21 years with the most common subtypes of sickle cell disease. Included articles focused on growth factors (platelet-derived growth factor), intra and extracellular brain proteins (glial fibrillary acidic protein, brain-derived neurotrophic factor), and inflammatory and coagulation markers (interleukin-1β, l -selectin, thrombospondin-1, erythrocyte, and platelet-derived microparticles). Positive findings include increases in plasma brain-derived neurotrophic factor and platelet-derived growth factor with elevated transcranial Dopplers velocities, increases in platelet-derived growth factor isoform AA with overt stroke, and increases in glial fibrillary acidic protein with acute brain injury. These promising potential neuro-biomarkers provide insight into pathophysiologic processes and clinical events, but their clinical utility is yet to be established. Additional proteomics research is needed, including broad-based proteomic discovery of plasma constituents and blood cell proteins, as well as urine and cerebrospinal fluid components, before, during and after neurological and developmental complications.     

Identification of proteins in human substantia nigra

Characterization of the human brain proteome is a critical area of research. While examination of the human cortex has provided some insight, very little is known about the proteome of the human midbrain, which demonstrates substantial loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in Parkinson's disease (PD). Therefore, characterization of this region is essential to a better understanding of the pathogenesis of PD. This dataset paper reports two separate studies, where human SNpc was collected from PD and control subjects and 1263 proteins were identified using MALDI-TOF/TOF as well as linear ion trap MS platforms. With gene ontology analysis, the proteins were categorized according to their biological processes, as well as cellular components. These data were also compared with previous proteomic characterization of the human frontal and temporal cortex, and cerebrospinal fluid to establish shared proteins of relevance. The present dataset is the most extensive survey of the human SNpc proteome, to date. Further characterization of the SNpc proteome will significantly facilitate our understanding of the function and expression of proteins involved in PD, as well as provide potential proteins that may be utilized as biomarkers.     

A nonlinear least-squares method for determining cerebrospinal fluid formation and absorption kinetics in pseudotumor cerebri

Data from 14 patients with benign intracranial hypertension (pseudotumor cerebri) have been analyzed using a nonlinear least-squares regression model which was developed and programmed from in-hospital microcomputer use. The method of analysis permits rapid estimation of cerebrospinal fluid (CSF) formation and absorption rates as functions of pressure in individual patients using data from constant-rate infusion manometrics. The analysis predicts that prednisone therapy in pseudotumor cerebri reduces resting CSF pressure by increasing CSF absorption at all intracranial pressures studied, and decreasing CSF formation at high pressures. This result is in accordance with evidence suggesting that impaired CSF absorption plays a major role in the pathogenesis of increased intracranial pressure in pseudotumor cerebri.     

Quantitative proteomic analysis of ovarian cancer cells identified mitochondrial proteins associated with Paclitaxel resistance

Paclitaxel has been widely used as an anti-mitotic agent in chemotherapy for a variety of cancers and adds substantial efficacy as the first-line chemotherapeutic regimen for ovarian cancers. However, the frequent occurrence of paclitaxel resistance limits its function in long-term management. Despite abundant clinical and cellular demonstration of paclitaxel resistant tumors, the molecular mechanisms leading to paclitaxel resistance are poorly understood. Using genomic approaches, we have previously identified an association between a BTB/POZ gene, Nac1, and paclitaxel resistance in ovarian cancer. The experiments presented here have applied multiple quantitative proteomic methods to identify protein changes associated with paclitaxel resistance and Nac1 function. The SKOV-3 ovarian serous carcinoma cell line, which has inducible expression of dominant negative Nac1, was used to determine the paclitaxel treatment associated changes in the presence and absence of functional Nac1. Quantitative proteomic analyses were performed using iTRAQ labeling and mass spectrometry. Two label-free quantitative proteomic methods: LC-MS and spectral count were used to increase confidence of proteomic quantification. A total of 1371 proteins were quantified by at least one of the quantitative proteomic methods. Candidate proteins related to paclitaxel and NAC1 function were identified in this study. Go analysis of the protein changes identified upon paclitaxel resistance revealed that cell component enrichment related to mitochondria. Moreover, tubulin and mitochondrial proteins were the major cellular components with changes associated with paclitaxel treatment. This suggests that mitochondria may play a role in paclitaxel resistance.     

Candidate antigens specifically detected by cerebrospinal fluid-IgG in oligoclonal IgG bands-positive multiple sclerosis patients

The aim of the present study was to detect antigenic proteins that react specifically with cerebrospinal fluid (CSF)-IgG from oligoclonal IgG bands (OB)-positive multiple sclerosis (MS) patients. To identify such antigenic proteins, we developed a rat brain proteome map using 2-DE and applied it to the immunoscreening of brain proteins that react with CSF-IgG but not with serum-IgG in OB-positive MS patients. After sequential MALDI-TOF mass spectrometry, eight proteins [two neuronal proteins (tubulin β-2 and γ enolase-2), HSP-1, Tpi-1 protein and cellular enzymes (creatine kinase, phosphopyruvate hydratase, triosephosphate isomerase and phosphoglycerate kinase-1)] were identified as candidate antigens in seven MS patients. Reactivity to tubulin was seen in Western blotting in four patients, and CSF-specific anti-tubulin IgG was detected in one patient. In addition, CSF-specific anti-gamma enolase IgG was found in another patient. These findings suggest that intrathecal immune responses may occur against a broad range of proteins in MS.     

Application of proteomic technologies to discover and identify biomarkers for periodontal diseases in gingival crevicular fluid: A review

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. In recent years, studies have shown a definite association between periodontal disease and other inflammatory conditions of the body. High-throughput analysis of proteins has become possible with the development of MS technology. This breakthrough in proteome technology enables comparative studies of comprehensive protein expression and identification of protein. In case of periodontal disease, proteome analysis using 2DE, as well as gel-free methods, has been reported. As a fluid lying in close proximity to periodontal tissue, the gingival crevicular fluid (GCF) is the principal target in the search for biomarkers of periodontal disease, because its protein composition may reflect the disease pathophysiology. Biochemical marker analysis of GCF is effective for objective diagnosis in the early and advanced stages of periodontal disease. Increasing numbers of recent reports have provided evidence that the proteomic approach is a promising tool for the discovery and identification of biochemical markers of periodontal disease. This search is of continuing interest in the field of experimental and clinical periodontal disease research. In this article, we summarize recent comprehensive proteomic studies aimed at discovering and identifying biomarkers of periodontal disease in GCF.     

Proteomic analysis in cerebrospinal fluid of patients with atypical nonketotic hyperglycinemia and pulmonary hypertension - A pilot study

A variant phenotype of nonketotic hyperglycinemia has been described by our group associated with pulmonary hypertension. The aim of this study is to investigate the cerebrospinal fluid proteomes to get an insight into this neurodegenerative process producing leukoencephalopathy with white matter spongiform degeneration. DIGE and MALDI-TOF-TOF analyses were performed to carry out the proteomic study of four patients against three normal controls and one additional control of a classical nonketotic hyperglycinemia. The differential proteomic analysis showed a displacement of some series of spots toward the acidic side. The shifted proteins showed a high degree of carbonylation and increased methionine sulfoxidation was found in cystatin C and in vitamin-D-binding protein. These findings in addition to the increase of serum malondialdehyde concentration provide evidence of an oxidative stress in the patients under study, which is probably systemic rather than mainly confined to the CNS. The similarities of our findings with those found in other neurodegenerative diseases suggest that oxidative damage is commonly involved in these pathologies. DIGE technology improves the 2-D PAGE differential analysis and it is suitable in proteomic studies with a small number of cases.     

Pre-analytical influence on the low molecular weight cerebrospinal fluid proteome

Cerebrospinal fluid (CSF) is a perfect source to search for new biomarkers to improve early diagnosis of neurological diseases. Standardization of pre‐analytical handling of the sample is, however, important to obtain acceptable analytical quality. In the present study, MALDI‐TOF MS was used to examine the influence of pre‐analytical sample procedures on the low molecular weight (MW) CSF proteome. Different storage conditions like temperature and duration or the addition of as little as 0.2 µL blood/mL neat CSF caused significant changes in the mass spectra. The performance of different types of MW cut‐off spin cartridges from different suppliers used to enrich the low MW CSF proteome showed great variance in cut‐off accuracy, stability and reproducibility. The described analytical method achieved a polypeptide discriminating limit of approximately 800 pM, two to three orders of magnitude lower than reported for plasma. Based on this study, we recommend that CSF is centrifuged immediately after sampling, prior to storage at –80ºC without addition of protease inhibitors. Guanidinium hydrochloride is preferred to break protein‐protein interactions. A spin cartridge with cut‐off limit above the intended analytical mass range is recommended. Our study contributes to the important task of developing standardized pre‐analytical protocols for the proteomic study of CSF.     

Shotgun MS proteomic analysis of bronchoalveolar lavage fluid in normal subjects

We provide a review of proteomic techniques used to characterize the bronchoalveolar lavage fluid (BALF) proteome of normal healthy subjects. Bronchoalveolar lavage (BAL) is the most common technique for sampling the components of the alveolar space. The proteomic techniques used to study normal BALF include protein separation by 2DE, whereby proteins were identified by comparison to a reference gel as well as high pressure liquid chromatography (HPLC)-MS/MS, also known as shotgun proteomics. We summarize recent progress using shotgun MS technologies to define the normal BALF proteome. Surprisingly, we find that despite advances in shotgun proteomic technologies over the course of the last 10 years, which have resulted in greater numbers of proteins being identified, the functional landscape of normal BALF proteome was similarly described by all methods examined.     

Proteomic analysis of serum, plasma, and lymph for the identification of biomarkers

Probably no topic has generated more excitement in the world of proteomics than the search for biomarkers. This excitement has been generated by two realities: the constant need for better biomarkers that can be used for disease diagnosis and prognosis, and the recent developments in proteomic technologies that are capable of scanning the individual proteins within varying complex clinical samples. Ideally a biomarker would be assayable from a noninvasively collected sample, therefore, much of the focus in proteomics has been on the analysis of biofluids such as serum, plasma, urine, cerebrospinal fluid, lymph, etc. While the discovery of biomarkers has been elusive, there have been many advances made in the understanding of the proteome content of various biofluids, and in the technologies used for their analysis, that continues to point the research community toward new methods for achieving the ultimate goal of identifying novel disease-specific biomarkers. In this review, we will describe and discuss many of the proteomic approaches taken in an attempt to find novel biomarkers in serum, plasma, and lymph.     

Functional regulation and proteomic characterization of human natural killer cells through recombinant human granulocyte‐colony stimulating factor treatment

Recombinant human granulocyte‐colony stimulating factor (rhG‐CSF) is the most common hematopoietic growth factor used for chemotherapy‐induced neutropenia or mobilization of stem cells. Natural killer (NK) cells are critical for host defense against infected cells and tumors. However, the cellular and molecular events of NK cells responding to rhG‐CSF remain unclear. Toward this end, we treated human NK cells with rhG‐CSF and assessed their cytotoxic function as well as proteomic characteristics. Unlike the other tested hematopoietic cytokines, rhG‐CSF decreased NK cell‐mediated cytotoxicity without affecting the viability of NK cells. The rhG‐CSF also reduced the production of nitric oxide and expression of inducible nitric oxide synthase in recombinant human interleukin (rhIL)‐2‐activated NK cells. By using cytokine array, rhG‐CSF reduced secretion of growth‐related oncogene‐α from NK cells. Intriguingly, rhG‐CSF did not affect production of various inflammatory cytokines [MCP‐1, IL‐6, tumor necrosis factor‐α (TNF‐α), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and IL‐8], which are markedly stimulated by rhGM‐CSF. Proteomic analysis of rhG‐CSF‐ and rhGM‐CSF‐treated NK cells uncovered unique proteins possibly involving rhG‐CSF effect. These proteins were classified into six groups as follows: glycolysis, apoptosis, cytotoxicity, inflammation, antigen processing and presentation, and the others. We conclude that rhG‐CSF may impair NK‐mediated cytotoxicity accompanied by alterations in protein expression profile distinct from that of rhGM‐CSF.     

Thymosin β4 is differentially expressed in the cerebrospinal fluid of Creutzfeldt‐Jakob disease patients: a MALDI‐TOF MS protein profiling study

MALDI‐TOF protein profiling analysis permits the detection of peptides and small proteins in complex protein mixtures with great accuracy. We applied this analysis to cerebrospinal fluid (CSF) from 15 patients affected by Creutzfeldt‐Jakob disease (CJD). We compared the levels of the normalized ion signals of 11 sporadic and 4 genetic CJD forms with those from ten healthy control subjects and eight non‐CJD relapsing‐remitting multiple sclerosis patients. In so doing, we detected 61 differentially expressed ion signals in CJD samples compared to controls. Among the 61 signals, 3 signals had significantly increased levels with high statistical significance (p <0.0001) and were located at 3238.3 m/z, 4963.7 m/z, and 8565.3 m/z. We characterized the 5.0 and 8.6 kDa proteins as thymosin β4 N‐acetylated and free ubiquitin, respectively, while the 3.2‐kDa peptide remained uncharacterized. Although elevated ubiquitin levels have previously been described in CJD, we have demonstrated for the first time the involvement of thymosin β4 in a neurodegenerative disease. To support the validity of thymosin β4 levels obtained by MALDI‐TOF analysis, an independent enzyme immunoassay analysis was performed. Moreover, a validation cohort consisting of CSF from three CJD patients, five healthy subjects, and six non‐CJD relapsing‐remitting multiple sclerosis patients was analyzed in a similar way, yielding superimposable results. We propose that thymosin β4 is a potential new candidate marker for the ante mortem diagnosis of CJD disease.     

Proteomics of human cerebrospinal fluid - the good, the bad, and the ugly

The development of MALDI ESI in the late 1980s has revolutionized the biological sciences and facilitated the emergence of a new discipline called proteomics. Application of proteomics to human cerebrospinal fluid (CSF) has greatly hastened the advancement of characterizing the CSF proteome as well as revealing novel protein biomarkers that are diagnostic of various neurological diseases. While impressive progressions have been made in this field, it has become increasingly clear that proteomics results generated by various laboratories are highly variable. The underlying issues are vast, including limitations and complications with heterogeneity of patients/testing subjects, experimental design, sample processing, as well as current proteomics technology. Accordingly, this review not only summarizes the current status of characterization of the human CSF proteome and biomarker discovery for major neurodegenerative disorders, i.e., Alzheimer's disease and Parkinson's disease, but also addresses a few essential caveats involved in several steps of CSF proteomics that may contribute to the variable/contradicting results reported by different laboratories. The potential future directions of CSF proteomics are also discussed with this analysis.