Top-down identification of endogenous peptides up to 9 kDa in cerebrospinal fluid and brain tissue by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry (original) (raw)
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Peptides in the Brain: Mass Spectrometry–Based Measurement Approaches and Challenges
Annual Review of Analytical Chemistry, 2008
The function and activity of almost every circuit in the human brain are modified by the signaling peptides (SPs) surrounding the neurons. As the complement of peptides can vary even in adjacent neurons and their physiological actions can occur over a broad range of concentrations, the required figures of merit for techniques to characterize SPs are surprisingly stringent. In this review, we describe the formation and catabolism of SPs and highlight a range of mass spectrometric techniques used to characterize SPs. Approaches that supply high chemical information content, direct tissue profiling, spatially resolved data, and temporal information on peptide release are also described. Because of advances in measurement technologies, our knowledge of SPs has greatly increased over the last decade, and SP discoveries will continue as the capabilities of modern measurement approaches improve.
Mass Spectrometry of Peptides in Neuroscience
Peptides, 1998
This review focuses on the contributions of modern mass spectrometry to neuropeptide research. An introduction to newer mass spectrometric techniques is provided. Also, the use of mass spectrometry in combination with high-resolution separation techniques for neuropeptide identification in biological samples is illustrated. The amino acid sequence information that is important for the identification and analysis of known, novel, or chemically modified neuropeptides may be obtained using mass spectrometric techniques. Because mass spectrometry techniques can be used to reflect the dynamic properties associated with neuropeptide processing in biological systems, they may be used in the future to monitor peptide profiles within organisms in response to environmental challenges such as disease and stress.
Methods in molecular biology (Clifton, N.J.), 2000
Recent developments in technology and instrumentation have made mass spectrometry the method of choice for the identification of gel-separated proteins using rapidly growing sequence databases (1). Proteins with a full-length sequence present in a database can be identified with high certainty and high throughput using the highly accurate masses obtained by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry peptide mapping (2). Simple protein mixtures can also be deciphered by MALDI peptide mapping (3) and the entire identification process, starting from in-gel digestion (4) and finishing with acquisition of mass spectra and database search, can be automated (5). Only 1-3% of a total digest are consumed for MALDI analysis even if the protein of interest is present on a gel in a subpicomole amount. If no conclusive identification is achieved by MALDI peptide mapping, the remaining protein digest can be analyzed by nanoelectrospray tandem mass spectrometry (Nano ES-MS/MS) (6). Nano ES-MS/MS produces data that allow highly specific database searches so that proteins that are only partially present in a database, or relevant clones in an EST database, can be identified (7). It is important to point out that there is no need to determine the complete sequence of peptides in order to search a database-a short sequence stretch consisting of three to four amino acid residues provides enough search specificity when combined with the mass of the intact peptide and the masses of corresponding
Journal of Proteome Research, 2009
In recent years, mass spectrometry (MS) based techniques have made their entrance in the analysis of endogenous peptides extracted from nervous tissue. In this study, we introduce a novel peptide extraction procedure using 8 M urea, next to the more established extraction method that uses acetic acid. The extracted peptide mixtures are analyzed by both high-resolution nanoLC MS/MS using collision induced dissociation (CID) on an LTQ-Orbitrap and nanoLC electron transfer induced dissociation (ETD) on a linear ion trap. The combined use of the two extraction methods significantly increased the yield of identified endogenous neuropeptides. The multiplexed use of high mass accuracy mass spectrometry and the ETD fragmentation technique further increased the yield and confidence of peptide identifications. Furthermore, reduction of disulfide bridges during sample preparation was essential in the identification of several endogenous peptides containing cysteine disulfide bonds. Through this study, we identified in total 142 peptides in extracts of the mouse pituitary tissue, whereby 43 uniquely in the urea extract and 11 uniquely in the acetic acid extract. A large number of detected endogenous peptides were reported previously, but we confidently identified 22 unreported peptides that possess characteristics of endogenous peptides and are thus interesting targets to be explored further.
PROTEOMICS – CLINICAL APPLICATIONS, 2007
Cerebrospinal fluid (CSF) perfuses the brain and spinal cord. CSF contains peptides and proteins important for brain physiology and potentially also relevant to brain pathology. High-throughput endogeneous peptide profiling by MS is an emerging approach for disease diagnosis and biomarker discovery. A magnetic bead-based method for off-line serum peptide capture coupled to MALDI-TOF-MS has been introduced recently. In this study, we optimize the peptide capture method for profiling of CSF and investigate the effect of a number of preanalytical variables. The CSF profiles contain ,100 reliably detected peptides at m/z 800-4000 with reproducible ion intensities (average 7% CV). The investigated preanalytical variables include: time at room temperature (RT) before storage, storage temperature, freeze-thawing cycles, and blood contamination. The CSF peptidome (,20 kDa) is relatively stable and can withstand a few hours at RT and several freeze-thaw cycles. Several peptides sensitive to storage at 2207C, including Cystatin C, were assigned based on mass or identified by MS/MS. Hemoglobin a and b chains were detected in blood contaminated samples, at levels invisible to the eye (0.01%). These peptides may be used for quality control in a MALDI-TOF-MS screening strategy to select high quality samples for indepth proteomics analysis in disease studies.
Journal of Separation Science, 2002
High performance capillary electrophoresis (HPCE) in an off-line combination with electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry (MS) has been developed for separation, identification, and compositional analysis of peptides. Two mixtures of peptides from an extracellular matrix have been separated and profiled by CE with UV detection. Introduction of ammonium formate in aqueous/ methanol solution at low pH values provided suitable buffer conditions for a good separation of components and reproducibility of experiments. The analytes were identified by subjecting the CE-collected fractions to (+)nanoESI-QTOF-MS and tandem MS with low energy collision induced dissociation (CID). CE/MS analysis of the mixture generated by peptide hydrolysis has been designed to allow both determination of the peptide amino acid composition and ladder sequencing. The strategy presented here could represent a useful preliminary method to serve as a basis for online experiments and for further analysis of proteins and their full structural elucidation.
Analytica chimica acta, 2014
Peptides in urine are excreted by kidney from the blood and tissues, which are composed of a large amount of hormones, cytokines, regulatory factors and the metabolized fragments of proteins. The peptide distribution in urine will reflect the physiological and pathophysiological processes in body. In past, limited information was reported about the composition of the peptides in urine. One possible reason is that the peptides in urine are fairly low abundant and there are high concentrations of salts and organic metabolites in the urine. In this report, we extracted the peptides from human urine by highly ordered mesoporous silica particles with the pore size of 2 nm, which will exclude the high molecular weight proteins over 12 kDa. The extracted peptides were then separated into fractions according to their molecular weight by size exclusion chromatography. Each of the fractions was further analyzed by MALDI-TOF MS and μRPLC-MS/MS. Totally, 193 peptides were identified by two-dime...