Peptide profiling of cerebrospinal fluid by mass spectrometry

The search for biomarkers is driven by the increasing clinical importance of early diagnosis. Reliable biomarkers can also assist in directing therapy, monitoring disease activity and the efficacy of treatment. In addition, the discovery of novel biomarkers might provide clues to the pathogenesis of a disease. The dynamic range of protein concentrations in body fluids exceeds 10 orders of magnitude. These huge differences in concentrations complicate the detection of proteins with low expression levels. Since all classical biomarkers have low expression levels (e.g., prostate-specific antigen: 2–4 µg/l; and CA125: 20–35 U/ml), new developments with respect to identification and validation techniques of the low-abundance proteins are required. This review will discuss the current status of profiling cerebrospinal fluid using mass spectrometry-based techniques, and new developments in this area.     

Peptide profiling of cerebrospinal fluid by mass spectrometry

The search for biomarkers is driven by the increasing clinical importance of early diagnosis. Reliable biomarkers can also assist in directing therapy, monitoring disease activity and the efficacy of treatment. In addition, the discovery of novel biomarkers might provide clues to the pathogenesis of a disease. The dynamic range of protein concentrations in body fluids exceeds 10 orders of magnitude. These huge differences in concentrations complicate the detection of proteins with low expression levels. Since all classical biomarkers have low expression levels (e.g., prostate-specific antigen: 2-4 microg/l; and CA125: 20-35 U/ml), new developments with respect to identification and validation techniques of the low-abundance proteins are required. This review will discuss the current status of profiling cerebrospinal fluid using mass spectrometry-based techniques, and new developments in this area.     

Characterization of tau in cerebrospinal fluid using mass spectrometry

The neurodegenerative disorder Alzheimer's disease (AD) is the most common cause of dementia in the elderly. The presence of neurofibrillary tangles, consisting of hyperphosphorylated tau protein, is one of the major neuropathologic characteristics of the disease, making this protein an attractive biomarker for AD and a possible target for therapy. Here, we describe an optimized immunoprecipitation mass spectrometry method that enables, for the first time, detailed characterization of tau in human cerebrospinal fluid. The identities of putative tau fragments were confirmed using nanoflow liquid chromatography and tandem mass spectrometry. Nineteen tryptic fragments of tau were detected, of which 16 are found in all tau isoforms while 3 represented unique tau isoforms. These results pave the way for clinical CSF studies on the tauopathies.     

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE.     

Prefractionation of proteome by liquid isoelectric focusing prior to two-dimensional liquid chromatography mass spectrometric identification

Due to the complexity of proteomes, developing methods of sample fractionation, separation, concentration, and detection have become urgent to the identification of large numbers of proteins, as well as the acquisition of those proteins in low abundance. In this work, liquid isoelectric focusing (LIEF) combined with 2D-LC-MS/MS was applied to the proteome of Saccharomyces cerevisiae. This yielded a total of 1795 proteins that were detected and identified by 30 fractions of protein prefractionation. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis without protein fractionation. LIEF-2D-LC-MS/MS also produced improved resolution of low-abundance proteins. Furthermore, we analyzed the characteristics of proteins obtained by LIEF-2D-LC-MS/MS. 1103 proteins with CAI under 0.2 were identified, allowing us to specifically obtain detailed biochemical information on these kind proteins. It was observed that LIEF-2D-LC-MS/MS is useful for large-scale proteome analysis and may be specifically applied to systems with wide dynamic ranges.     

Multiple sclerosis-related proteins identified in cerebrospinal fluid by advanced mass spectrometry

A total of 164 cerebrospinal fluid (CSF) samples taken from neurological patients were classed into four groups according to the clinical diagnosis: multiple sclerosis (MScl, n = 44), clinically isolated syndrome of demyelination (CIS, n = 40), other inflammatory neurological disease (OIND, n = 26) and other neurological disease (OND, n = 54). After tryptic digestion, the samples were measured by MALDI-TOF MS. Spectra were analyzed using the R-project software package, in which a peak detection algorithm was developed. Subsequently, the peak lists were compared based on ranked data (non-parametric). Significant differences were observed in the comparisons of MScl vs. OND and CIS vs. OND. The comparisons of MScl vs. OIND, and CIS vs. OIND showed fewer significant differences. No significant differences were found in comparisons MScl vs. CIS and OIND vs. OND. MScl and CIS had strikingly similar profiles, probably a reflection of common pathological mechanisms. Three differentially expressed proteins in the comparison of MScl vs. OND were identified: chromogranin A, a potential marker for neurodegeneration; and two important factors in complement-mediated inflammatory reaction, clusterin and complement C3. CSF chromogranin A levels were confirmed to be significantly elevated in the MScl group using an ELISA.     

Quantitative determination of hederagenin in rat plasma and cerebrospinal fluid by ultra fast liquid chromatography-tandem mass spectrometry method

A rapid, sensitive and selective method was developed for the quantitative determination of hederagenin in rat plasma and cerebrospinal fluid (CSF) by ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS). It has been successfully applied in a pharmacokinetic study of hederagenin in the central nervous system (CNS). Sample pretreatment involved a simple protein precipitation with methanol and a one-step extraction with ethyl acetate. Separation was carried out in a Shim-pack XR-ODS II (75 mm × 2.0 mm, i.d., 2.1 μm) column with gradient elution at a flow rate of 0.35 mL/min. The mobile phase was 5mM ammonium acetate and acetonitrile. Detection was performed in a triple-quadruple tandem mass spectrometer by multiple-reaction-monitoring mode via electrospray ionization. A linear calibration curve for hederagenin was obtained over a concentration range of 0.406 (lower limit of quantification, LLOQ) to 203 ng/mL (r2 > 0.99) for both plasma and CSF. The intra-day and inter-day precision (relative standard deviation, RSD) values were less than 15%. At all quality control (QC) levels, the accuracy (relative error, RE) was within -9.0% and 11.1% for plasma and CSF, respectively. The pharmacokinetics results indicated that hederagenin could pass through the blood-brain barrier. This UFLC-MS/MS method demonstrates higher sensitivity and sample throughput than previous methods. It was also successfully applied to the pharmacokinetic study of hederagenin following oral administration of Fructus akebiae extract in rats.     

Identification of neuropeptide FF-related peptides in human cerebrospinal fluid by mass spectrometry

Several neuropeptide FF (NPFF)-related peptides, known as modulators of the opioid system, have been previously characterized in bovine and rodent brain. Reverse-phase high pressure liquid chromatography (HPLC) fractions of a human with normal pressure hydrocephalus cerebrospinal fluid (CSF), co-migrating with NPFF-related synthetic peptides, were characterized by capillary HPLC coupled on-line to nanospray ion trap tandem mass spectrometry. Two peptides present in the pro-NPFF(A) precursor, NPAF (AGEGLNSQFWSLAAPQRF-NH2) and NPSF (SLAAPQRF-NH2), were identified. The monitoring of NPFF-related peptides in human CSF can be helpful to understand their roles in pain sensitivity.     

Understanding glycoprotein structural heterogeneity and interactions: Insights from native mass spectrometry

Protein glycosylation is critical since it connects complex metabolic pathways to diverse proteoforms, fine-tunes protein structures and exerts biological functions. Aberrant glycosylation on the other hand is associated with many diseases, including cancers, inflammation and metabolic disorders. By resolving monosaccharide residues on intact glycoprotein complexes, native mass spectrometry can shed light on glycan heterogeneity, glycoprotein structure and molecular recognition. Here, we focus on the two most prevalent forms of glycosylation, namely N- and O- linked, and discuss recent progress in native mass spectrometry for elucidating glycoprotein structural heterogeneity and relating specific glycan repertoires to glycoprotein interactions.     

MALDI-TOF mass spectrometry analysis of cerebrospinal fluid tryptic peptide profiles to diagnose leptomeningeal metastases in patients with breast cancer

Leptomeningeal metastasis (LM) is a devastating complication that occurs in 5% of patients with breast cancer. Early diagnosis and initiation of treatment are essential to prevent neurological deterioration. However, early diagnosis of LM remains challenging because 25% of cerebrospinal fluid (CSF) samples produce false-negative results at first cytological examination. We developed a new, MS-based method to investigate the protein expression patterns present in the CSF from patients with breast cancer with and without LM. CSF samples from 106 patients with active breast cancer (54 with LM and 52 without LM) and 45 control subjects were digested with trypsin. The resulting peptides were measured by MALDI-TOF MS. Then, the mass spectra were analyzed and compared between patient groups using newly developed bioinformatics tools. A total of 895 possible peak positions was detected, and 164 of these peaks discriminated between the patient groups (Kruskal-Wallis, p<0.01). The discriminatory masses were clustered, and a classifier was built to distinguish patients with breast cancer with and without LM. After bootstrap validation, the classifier had a maximum accuracy of 77% with a sensitivity of 79% and a specificity of 76%. Direct MALDI-TOF analysis of tryptic digests of CSF gives reproducible peptide profiles that can assist in diagnosing LM in patients with breast cancer. The same method can be used to develop diagnostic assays for other neurological disorders.     

Porous graphitic carbon chromatography-tandem mass spectrometry for the study of isoprostanes in human cerebrospinal fluid

F2-isoprostanes are produced by the non-enzymatic peroxidation of arachidonic acid in membrane phospholipids. This paper describes a new method for the determination of all four classes of F2-isoprostanes in human cerebrospinal fluid (CSF) involving separation on a 1 mm x 150 mm porous graphitic carbon (PGC) column and detection by triple quadrupole mass spectrometry in negative-ion electrospray mode. The sample pre-treatment consisted of an ultrafiltration step, following which 300 microl of CSF sample could be injected directly onto a 1 mm x 10 mm PGC guard column functioning as a trap for the analytes. The loading solvent was Milli-Q water at 125 microl/min. After 3 min, the sample was switched into the separation column. The F2-isoprostanes were separated in 20 min using a linear solvent gradient comprising water, methanol, acetonitrile and ammonium hydroxide at a pH of 9.5 and a flow of 50 microl/min The limit of detection (calculated as 3S/N) was approximately 40 pM (14 pg/ml). The assay was linear within the examined range (18-450 pg/ml), using CSF spiked with iPF2alpha-III standard (r(2)>0.995). Repeatability data were calculated for CSF spiked to 90 pg/ml and the relative standard deviation (RSD) obtained was 3% (n=6).     

Quantitative analysis of amyloid-beta peptides in cerebrospinal fluid using immunoprecipitation and MALDI-Tof mass spectrometry.

Immunoprecipitation (IP) combined with matrix-assisted laser desorption ionization (MALDI) time of flight (Tof) mass spectrometry has been used to develop quantitative assays for amyloid-beta (Abeta) peptides in cerebrospinal fluid (CSF). Inclusion of (15)N labelled standard peptides allows for absolute quantification of multiple Abeta isoforms in individual samples. Characterization of variability associated with all steps of the assay indicated that the IP step is the single largest contributor to overall variability. Optimization of the assay resulted in overall coefficient of variation 相似文献   

Structural analysis of 2D-gel-separated glycoproteins from human cerebrospinal fluid by tandem high-resolution mass spectrometry

The feasibility of global glycoprotein analysis by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry is demonstrated. Combined 2D gel glycoprotein separation and visualization, in-gel digestion, and accurate (<10 ppm) mass measurement allowed identification of human glycoproteins and revealed differences in glycosylation. IRMPD obviates the need for glycan release, which prevents sample dispersal, and allows the assignment of glycan structures to specific sites of N-glycosylation.     

Determination of baicalin in rat cerebrospinal fluid and blood using microdialysis coupled with ultra-performance liquid chromatography-tandem mass spectrometry

An in vivo microdialysis sampling method coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed for continuous simultaneous monitoring of unbound baicalin in rat blood and brain. Microdialysis probes were inserted into the jugular vein and brain cerebrospinal fluid (CSF) of Sprague-Dawley rats then, following administration of baicalin at doses of 24mg/kg via the candal vein, samples were collected every 20min and injected directly into the UPLC-MS/MS system. In vitro recoveries of the probes were 19.26% and 18.38%, while in vivo recoveries of the probes were 15.0% and 17.52% for blood and brain, respectively. This improved method offers a rapid quantitative procedure for the determination of baicalin with a retention time of only 1.6min. The lower limit of quantification (LLOQ) and the lower limit of detection (LLOD) based on a signal-to-noise ratio of 5 were 2.37 and 0.1ng/ml for anticoagulant citrate dextrose (ACD) solution, and 1.185 and 0.3ng/ml for artificial cerebrospinal fluid (aCSF), respectively. The pharmacokinetics results indicated that baicalin could pass through the blood-brain barrier (BBB) and was detectable in brain dialysate. These in vivo microdialysis-based measurements provide a technique for simple sampling and rapid sensitive analysis of unbound baicalin in rat blood and CSF and for further application in pharmacokinetic studies.     

High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry

As the number of fully sequenced animal genomes and the performance of advanced mass spectrometry-based proteomics techniques are continuously improving, there is now a great opportunity to increase the knowledge of various animal proteomes. This research area is further stimulated by a growing interest from veterinary medicine and the pharmaceutical industry. Cerebrospinal fluid (CSF) is a good source for better understanding of diseases related to the central nervous system, both in humans and other animals.In this study, four high-abundant protein depletion columns, developed for human or rat serum, were evaluated for dog CSF. For the analysis, a shotgun proteomics approach, based on nanoLC-LTQ Orbitrap MS/MS, was applied. All the selected approaches were shown to deplete dog CSF with different success. It was demonstrated that the columns significantly improved the coverage of the detected dog CSF proteome. An antibody-based column showed the best performance, in terms of efficiency, repeatability and the number of proteins detected in the sample. In total 983 proteins were detected. Of those, 801 proteins were stated as uncharacterized in the UniProt database. To the best of our knowledge, this is the so far largest number of proteins reported for dog CSF in one single study.     

Liquid chromatography-tandem mass spectrometry method for determination of N-acetylglucosamine concentration in human plasma

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for quantification of N-acetylglucosamine in human plasma. Plasma samples were pretreated with acetonitrile for protein precipitation. The chromatographic separation was performed on Hypersil Silica column (150mmx2mm, 5microm). The deprotonated analyte ion was detected in negative ionization mode by multiple reaction monitoring mode. The mass transition pairs of m/z 220.3-->118.9 and m/z 226.4-->123.2 were used to detect N-acetylglucosamine and internal standard 13C6-N-acetylglucosamine, respectively. The assay exhibited a linear range from 20 to 1280ng/ml for N-acetylglucosamine in human plasma. Acceptable precision and accuracy were obtained for concentrations of the calibration standard and quality control. The validated method was successfully applied to analyze human plasma samples in a pharmacokinetic study.     

生物质谱技术在糖蛋白结构分析中的应用

生物质谱包括基质辅助激光解吸附飞行时间质谱及电喷雾质谱被广泛应用于生物样品如多肽、蛋白质及核酸的分析,由于这种具有软电离方式的质谱具有极高的灵敏度及准确度,目前也被成功地用于糖蛋白的结构分析,与普通的化学方法相比,质谱法快速、简单,结合网上数据库检索、凝集素亲和提取、二维凝胶电泳以及靶上直接酶切等新方法,可以提供糖蛋白的一级结构乃至高级结构的信息。     

A combined dataset of human cerebrospinal fluid proteins identified by multi-dimensional chromatography and tandem mass spectrometry

Human cerebrospinal fluid (CSF) is an important source for studying protein biomarkers of age-related neurodegenerative diseases. Before characterizing biomarkers unique to each disease, it is necessary to categorize CSF proteins systematically and extensively. However, the enormous complexity, great dynamic range of protein concentrations, and tremendous protein heterogeneity due to post-translational modification of CSF create significant challenges to the existing proteomics technologies for an in-depth, nonbiased profiling of the human CSF proteome. To circumvent these difficulties, in the last few years, we have utilized several different separation methodologies and mass spectrometric platforms that greatly enhanced the identification coverage and the depth of protein profiling of CSF to characterize CSF proteome. In total, 2594 proteins were identified in well-characterized pooled human CSF samples using stringent proteomics criteria. This report summarizes our efforts to comprehensively characterize the human CSF proteome to date.     

Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry

A new hydrophilic interaction chromatography (HILIC) column packed with amide 1.7 μm sorbent was applied to the characterization of glycoprotein digests. Due to the impact of the hydrophilic carbohydrate moiety, glycopeptides were more strongly retained on the column and separated from the remaining nonglycosylated peptides present in the digest. The glycoforms of the same parent peptide were also chromatographically resolved and analyzed using ultraviolet and mass spectrometry detectors. The HILIC method was applied to glyco-profiling of a therapeutic monoclonal antibody and proteins with several N-linked and O-linked glycosylation sites. For characterization of complex proteins with multiple glycosylation sites we utilized 2D LC, where RP separation dimension was used for isolation of glycopeptides and HILIC for resolution of peptide glycoforms. The analysis of site-specific glycan microheterogeneity was illustrated for the CD44 fusion protein.     

Gas-liquid chromatography and mass spectrometry of methylated and acetylated methyl glycosides. Application to the structural analysis of glycoprotein glycans

The mass spectra of 52 partially methylated and acetylated methyl glycosides of galactose, mannose, glucose, and N-acetylglucosamine have been determined. Each derivative was identified on the basis of its gas-liquid chromatography retention time and mass spectra. The analysis of methyl ethers obtained by methanolysis of fully methylated glycans of α1-acid glycoprotein is described as an application of the method.