Transitioning from cerebrospinal fluid to blood tests to facilitate diagnosis and disease monitoring in Alzheimer's disease.

Alzheimer's disease (AD) is increasingly prevalent worldwide, and disease-modifying treatments may soon be at hand; hence, now, more than ever, there is a need to develop techniques that allow earlier and more secure diagnosis. Current biomarker-based guidelines for AD diagnosis, which have replaced the historical symptom-based guidelines, rely heavily on neuroimaging and cerebrospinal fluid (CSF) sampling. While these have greatly improved the diagnostic accuracy of AD pathophysiology, they are less practical for application in primary care, population-based and epidemiological settings, or where resources are limited. In contrast, blood is a more accessible and cost-effective source of biomarkers in AD. In this review paper, using the recently proposed amyloid, tau and neurodegeneration [AT(N)] criteria as a framework towards a biological definition of AD, we discuss recent advances in biofluid-based biomarkers, with a particular emphasis on those with potential to be translated into blood-based biomarkers. We provide an overview of the research conducted both in CSF and in blood to draw conclusions on biomarkers that show promise. Given the evidence collated in this review, plasma neurofilament light chain (N) and phosphorylated tau (p-tau; T) show particular potential for translation into clinical practice. However, p-tau requires more comparisons to be conducted between its various epitopes before conclusions can be made as to which one most robustly differentiates AD from non-AD dementias. Plasma amyloid beta (A) would prove invaluable as an early screening modality, but it requires very precise tests and robust pre-analytical protocols.

Identification of candidate cerebrospinal fluid biomarkers in parkinsonism using quantitative proteomics.

INTRODUCTION: Neurodegenerative parkinsonian syndromes have significant clinical and pathological overlap, making early diagnosis difficult. Cerebrospinal fluid (CSF) biomarkers may aid the differentiation of these disorders, but other than α-synuclein and neurofilament light chain protein, which have limited diagnostic power, specific protein biomarkers remain elusive. OBJECTIVES: To study disease mechanisms and identify possible CSF diagnostic biomarkers through discovery proteomics, which discriminate parkinsonian syndromes from healthy controls. METHODS: CSF was collected consecutively from 134 participants; Parkinson's disease (n = 26), atypical parkinsonian syndromes (n = 78, including progressive supranuclear palsy (n = 36), multiple system atrophy (n = 28), corticobasal syndrome (n = 14)), and elderly healthy controls (n = 30). Participants were divided into a discovery and a validation set for analysis. The samples were subjected to tryptic digestion, followed by liquid chromatography-mass spectrometry analysis for identification and relative quantification by isobaric labelling. Candidate protein biomarkers were identified based on the relative abundances of the identified tryptic peptides. Their predictive performance was evaluated by analysis of the validation set. RESULTS: 79 tryptic peptides, derived from 26 proteins were found to differ significantly between atypical parkinsonism patients and controls. They included acute phase/inflammatory markers and neuronal/synaptic markers, which were respectively increased or decreased in atypical parkinsonism, while their levels in PD subjects were intermediate between controls and atypical parkinsonism. CONCLUSION: Using an unbiased proteomic approach, proteins were identified that were able to differentiate atypical parkinsonian syndrome patients from healthy controls. Our study indicates that markers that may reflect neuronal function and/or plasticity, such as the amyloid precursor protein, and inflammatory markers may hold future promise as candidate biomarkers in parkinsonism.

Large-gel two-dimensional electrophoresis-matrix assisted laser desorption/ionization-time of flight-mass spectrometry: an analytical challenge for studying complex protein mixtures.

The large-gel two-dimensional electrophoresis (2-DE) technique, developed by Klose and co-workers over the past 25 years, provides the resolving power necessary to separate crude proteome extracts of higher eukaryotes. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) provides the sample throughput necessary to identify thousands of different protein species in an adequate time period. Spot excision, in situ proteolysis, and extraction of the cleavage products from the gel matrix, peptide purification and concentration as well as the mass spectrometric sample preparation are the crucial steps that interface the two analytical techniques. Today, these routines and not the mass spectrometric instrumentation determine how many protein digests can be analyzed per day per instrument. The present paper focuses on this analytical interface and reports on an integrated protocol and technology developed in our laboratory. Automated identification of proteins in sequence databases by mass spectrometric peptide mapping requires a powerful search engine that makes full use of the information contained in the experimental data, and scores the search results accordingly. This challenge is heading a second part of the paper.

Identification of nuclei associated proteins by 2D-gel electrophoresis and mass spectrometry.

In clinical neuroscience as well as in many other clinical disciplines, the completion of the human genome project offers a new possibility to identify and localize the products of the genes, the proteins. Nuclear proteins are synthesized in the cytoplasm and imported into the nucleus by multiple pathways. The mechanisms by which nuclear accumulation of different molecular species occur are unclear but it is apparent that changes in the cellular and molecular events associated with the accumulation of nuclear proteins sometimes precedes transformation of cells into diseased states. The significance of the accumulation and the operation of nuclear proteins remain to be elucidated in detail. Such knowledge will play a key role in the understanding of the regulation of transcription and its disturbances in several of our most devastating diseases. In this paper we present a strategy to identify nuclear associated proteins in small samples by using two-dimensional electrophoresis and mass spectrometry. We have used human blood lymphocytes as a model, but the method should be rather general for any kind of tissue. Twenty two proteins were randomly chosen, and of these 18 proteins were identified by database searching of mass spectrometric data, obtained from in-gel tryptic digests of the spots. Thirteen proteins recently described with nuclear localization and function were identified, and five proteins; calgranulin B, glyceraldehyde-3-phosphate dehydrogenase (G3P2), a TATA-binding protein (ATBP), tubulin beta chain and moesin were also identified as being nuclear associated. The presented data clearly shows of the great role of two-dimensional gel electrophoresis and modern mass spectrometry in the excavation of the protein patterns on the subcellular level, and the ability to use small samples well suited for clinical screening.

A CE-MALDI interface based on the use of prestructured sample supports.

We have developed an off-line coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) based on CE fraction collection onto prestructured MALDI sample supports. Analyte carryover and detection sensitivity were investigated using a standard peptide mixture. Low femtomole amounts were detected, and no noticeable carryover was discovered. The performance of the method was evaluated with a mixture of tryptic digests of proteins from a human fetal brain cDNA expression library. The total number of identified peptides was increased from 47 to 211 when the CE-MALDI interface was used compared to direct MALDI-MS analysis. Sequence coverage with CE-MALDI was in the 25-60% range for the different proteins, corresponding to an increase of 1.3-4.9 times relative to that obtained with MALDI-MS of the crude mixture. Fractionation of sample components also facilitated protein identification by MALDI postsource decay analysis. Our initial results suggest this CE-MALDI interface can be used for the analysis of complex peptide mixtures isolated from biological tissues.

Arginine vasopressin in the cytoplasm and nuclear fraction of lymphocytes from healthy donors and patients with depression or schizophrenia.

We investigated whether cytoplasmic or nuclear extracts of human peripheral blood lymphocytes contain AVP in samples from healthy controls and patients diagnosed as depressed or schizophrenic. Both the cytoplasmic and nuclear extracts contained AVP as determined by radioimmunoassay. AVP and other peptides were detected in the purified samples by matrix-assisted laser desorption/ionization time of flight mass spectrometry. It is the first time that AVP has been characterized in human lymphocytes of patients with depression or schizophrenia. This finding demonstrates the presence of another important component within the potential regulatory loop between immune and neuro-endocrine tissues.

Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics.

We present a new MALD1 sample preparation technique for peptide analysis using the matrix alpha-cyano-4-hydroxy-cinnamic acid (CHCA) and prestructured sample supports. The preparation integrates sample purification, based on the affinity of microcrystalline CHCA for peptides, thereby simplifying the analysis of crude peptide mixtures. Enzymatic digests can thus be prepared directly, without preceding purification. Prepared samples are homogeneous, facilitating automatic spectra acquisition. This method allows preparation of large numbers of samples with little effort and without the need for automation. These features make the described preparation suitable for cost-efficient high-throughput protein identification. Performance of the sample preparation is demonstrated with in situ proteolytic digests of human brain proteins separated by two-dimensional gel electrophoresis.

Detection and quantification of neurotensin in human brain tissue by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A method was developed for mass spectrometric detection of neurotensin (NT)-like immunoreactivity and quantification of NT in human brain tissue. The method is based on immunoprecipitation followed by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The identity of the major component of the immunoprecipitates as neurotensin was confirmed by fragment ion analysis on an electrospray ionization quadrupole time-of-flight instrument. MALDI-TOF-MS quantification of NT was achieved using stable-isotope-labeled NT as the internal standard, yielding an error of less than 5%. The method allowed detection of low-femtomole amounts of NT, staring from low-milligram amounts of lyophilized brain tissue. In addition to NT, several other peptides were detected in the purified samples, most of which, according to their molecular masses, corresponded to fragments of NT. The method is demonstrated with quantification of NT from human hypothalamus tissue, and a comparison is made with results obtained from competitive radioimmunoassay.

Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry.

A simple reversed-phase nano-column purification and sample preparation technique is described, which markedly improves the mass spectrometric analysis of complex and contaminated peptide mixtures by matrix-assisted laser desorption/ionization (MALDI). The method is simple, fast and utilizes only low-cost disposables. After loading the sample on the column and a subsequent washing step, the analyte molecules are eluted with 50-100 nl of matrix solution directly on to the MALDI/MS target. The washing step ensures removal of a wide range of contaminants. The small bed volume of the column allows efficient sample concentration and the elution process yields very small sample spots. This simplifies the analysis and minimizes discrimination effects due to sample heterogeneity, because the desorption/ionization laser simultaneously irradiates a large portion of the sample. Taken together, these features of the method significantly improve the sensitivity for MALDI/MS analysis of contaminated peptide samples compared with the commonly used sample preparation procedures. This is demonstrated with in-gel tryptic digests of proteins from human brain that were separated by 2D gel electrophoresis. Furthermore, it is shown that with this method 2,5-dihydroxybenzoic acid (DHB) acts as an efficient matrix for peptide mapping. Both detection sensitivity and sequence coverage are comparable to those obtained with the currently preferred matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). The higher stability of peptide ions generated with DHB compared with CHCA is advantageous when analyzing fragile sample molecules. Therefore, the method described here is also of interest for the use of Fourier transform ion cyclotron resonance (FT-ICR) or ion-trap mass analyzers.

Use of vapor-phase acid hydrolysis for mass spectrometric peptide mapping and protein identification.

A method for mass spectrometric peptide mapping was developed, based on hydrolysis of a solid protein by acid vapor followed by mass spectrometric analysis of the cleavage products. The method is applicable to lyophilized samples as well as proteins present in gels after separation by SDS-PAGE. The cleavage specificity was established using a number of standard proteins. Three different types of cleavages were observed: specific internal backbone cleavages at Asp, Ser, Thr, and Gly and N- and C-terminal sequence ladders. On the basis of the observed cleavage characteristics, a strategy for protein identification based on the peptide mass maps was developed. The identification strategy utilizes the specific internal backbone cleavages as well as the partial sequence information, obtained from the sequence ladders.