High-resolution peptide mapping of cerebrospinal fluid: a novel concept for diagnosis and research in central nervous system diseases (original) (raw)
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Special feature: Perspective. Mass spectrometric peptide and protein charting
Journal of Mass Spectrometry, 1995
Six years after coining the term 'mass spectrometric (MS) peptide charting' for the component analysis of peptide mixtures in a whole tissue, body fluid, or an extract thereof, we offer our current perspective of this field. Matrixassisted laser desorptionlionization and electrospray ionization have replaced plasma desorption and fast atom bombardment as ionization methods of choice. At the same time, the upper mass range has been extended to now include most peptides and proteins of interest to research on cellcell communication. In addition to qualitative aspects, quantitative applications of MS charting have become important. In combination with new database search algorithms, on-line liquid chromatography-tandem mass spectrometry promises greater dividends from MS charting than are achievable with molecular mass matching alone. We discuss what is and is not yet possible, and consider foreseeable means for overcoming current limitations. Our intent is to encourage researchers in the biological and medical sciences to take advantage of this powerful methodology in their various fields of endeavor.
Molecular & cellular proteomics : MCP, 2010
Cerebrospinal fluid (CSF) is the biological fluid in closest contact with the brain and thus contains proteins of neural cell origin. Hence, CSF is a biochemical window into the brain and is particularly attractive for the search for biomarkers of neurological diseases. However, as in the case of other biological fluids, one of the main analytical challenges in proteomic characterization of the CSF is the very wide concentration range of proteins, largely exceeding the dynamic range of current analytical approaches. Here, we used the combinatorial peptide ligand library technology (ProteoMiner) to reduce the dynamic range of protein concentration in CSF and unmask previously undetected proteins by nano-LC-MS/MS analysis on an LTQ-Orbitrap mass spectrometer. This method was first applied on a large pool of CSF from different sources with the aim to better characterize the protein content of this fluid, especially for the low abundance components. We were able to identify 1212 protein...
Journal of Mass Spectrometry, 2005
Recent work on protein and peptide biomarker patterns revealed the difficulties in identifying their molecular components, which is indispensable for validation of the biological context. Cerebrospinal fluid and brain tissue are used as sources to discover new biomarkers, e.g. for neurodegenerative diseases. Many of these biomarker candidates are peptides with a molecular mass of <10 kDa. Their identification is favourably achieved with a 'top-down' approach, because this requires less purification and an enzymatic cleavage will often not yield enough specific fragments for successful database searches. Here, we describe an approach using quadrupole time-of-flight mass spectrometry (TOFMS) as a highly efficient mass spectrometric purification and identification tool after off-line decomplexation of biological samples by liquid chromatography. After initial peptidomic screening with matrix-assisted laser desorption/ionization (MALDI) TOFMS, the elution behaviour in chromatography and the exact molecular mass were used to locate the same signals in nanoelectrospray measurements. Most of the peaks detected in MALDI-TOFMS could be retrieved in nanoelectrospray quadrupole TOFMS. Suitable collision energies for informative fragment spectra were investigated for different parent ions, charge states and molecular masses. After collision-induced dissociation, the resulting fragmentation data of multiply charged ions can become much more complicated than those derived from tryptic peptide digests. However, the mass accuracy and resolution of quadrupole TOF instruments results in high-quality data suitable for determining peptide sequences. The protein precursor, proteolytic processing and post-translational modifications were identified by automated database searches. This is demonstrated by the exemplary identifications of thymosin beta-4 (5.0 kDa) and NPY (4.3 kDa) from rat hypothalamic tissue and ubiquitin (8.6 kDa) from human cerebrospinal fluid. The high data quality should also allow for de novo identification. This methodology is generally applicable for peptides up to a molecular mass of about 10 kDa from body fluids, tissues or other biological sources.
Improved resolution of human cerebrospinal fluid proteins on two-dimensional gels
Multiple Sclerosis Journal, 2003
Proteomics combines two-dimensional gel electro phoresis and peptide mass fingerprinting and can potentially identify a protein(s) unique to disease. Such proteins can be used either for diagnosis or may be relevant to the pathogenesis of disease. Because patients with multiple sclerosis (MS) have increased amounts of immunoglobulin (Ig) G in their cerebrospinal fluid (C SF) that is directed against an as yet unidentified protein, we are applying proteomics to MS C SF, studies that require optimal separation of proteins in human C SF. We found that recovery of proteins from C SF of MS patients was improved using ultrafiltration, rather than dialysis, for desalting. Resolution of these proteins was enhanced by aceto ne precipitatio n of desalted C SF before electrophoresis and by fractionation of C SF using C ibacron Blue sepharose affinity chromatography. Improved protein recovery and resolution will facilitate excision from gels for analysis by peptide mass fingerprinting.
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.
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 the American Society for Mass Spectrometry, 1999
We present a multidimensional approach to map the composition of complex peptide mixtures obtained as crude extract from biological liquids by (1) cation exchange chromatography and (2) subsequent microbore reversed-phase liquid chromatography and electrospray mass spectrometry coupling (LC-MS). Human hemofiltrate is an equivalent to blood and is used to obtain peptide material in large quantities from patients with chronic renal failure. The upper exclusion limit of the filtration membranes used results in a protein-free filtrate containing peptides in a range up to 20 ku. Using this unique peptide source, several thousand peptides were detected and an LC-MS data base of circulating human peptides was created. The search for known peptides by their molecular mass is a reliable method to guide peptide purification. (J Am Soc Mass Spectrom 1999, 10, 45-54)
Rapid high-sensitivity peptide mapping by liquid chromatography—mass spectrometry
Journal of Chromatography A, 1993
Toward a complete LC-MS mapping system for peptides and proteins, we have coupled a precision-flow microbore HPLC system to an electrospray single quadrupole mass spectrometer. The HPLC system allows fast separation of protein digests with UV detection at the low pmol level. A 2 pllmin portion (1:25) of the effluent is passed into a high-sensitivity electrospray MS system. The electrospray source allows for molecular mass associated ions (MH', MHZ', MH:', etc.) to be generated as well as collision induced dissociation of these ions before MS analysis. After LC-MS runs, with or without partial fragmentation, the data generated are largely interpreted by identification of predicted peptides, incompletely digested peptides, unusual peptide cleavages, and so on, using appropriate integrated software (PEPMAP, PEPMATCH). Examples of peptide mapping at the low pmol level using this integrated system will be shown (e.g., of the protein human growth hormone and of the glycoprotein, tissue plasminogen activator).