Expanding the cerebrospinal fluid endopeptidome

Biomarkers of neurodegenerative disorders are needed to assist in diagnosis, to monitor disease progression and therapeutic interventions, and to provide insight into disease mechanisms. One route to identify such biomarkers is by proteomic and peptidomic analysis of cerebrospinal fluid (CSF). In the current study, we performed an in‐depth analysis of the human CSF endopeptidome to establish an inventory that may serve as a basis for future targeted biomarker studies. High‐pH RP HPLC was employed for off‐line sample prefractionation followed by low‐pH nano‐LC‐MS analysis. Different software programs and scoring algorithms for peptide identification were employed and compared. A total of 18 031 endogenous peptides were identified at a FDR of 1%, increasing the number of known endogenous CSF peptides 10‐fold compared to previous studies. The peptides were derived from 2 053 proteins of which more than 60 have been linked to neurodegeneration. Notably, among the findings were six peptides derived from microtubule‐associated protein tau, three of which span the diagnostically interesting threonine‐181 (Tau‐F isoform). Also, 213 peptides from amyloid precursor protein were identified, 58 of which were partially or completely within the sequence of amyloid β 1–40/42, as well as 109 peptides from apolipoprotein E, spanning sequences that discriminate between the E2/E3/E4 isoforms of the protein.     

Peptidome analysis of human skim milk in term and preterm milk

The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. Due to the difficulties associated with detection technology of the peptides, the expression of the peptides present in human milk is not known widely. In recent years, peptidome analysis has received increasing attention. In this report, the analysis of endogenous peptides in human milk was done by mass spectrometry. A method was also developed by our researchers, which can be used in the extraction of peptide from human milk. Analysis of the extracts by LC–MS/MS resulted in the detection of 1000–3000 Da peptide-like features. Out of these, 419 peptides were identified by MS/MS. The identified peptides were found to originate from 34 proteins, of which several have been reported. Analysis of the peptides’ cleavage sites showed that the peptides are cleaved with regulations. This may reflect the protease activity and distribution in human body, and also represent the biological state of the tissue and provide a fresh source for biomarker discovery. Isotope dimethyl labeling analysis was also used to test the effects of premature delivery on milk protein composition in this study. Differences in peptides expression between breast milk in term milk (38–41 weeks gestation) and preterm milk (28–32 weeks gestation) were investigated in this study. 41 Peptides in these two groups were found expressed differently. 23 Peptides were present at higher levels in preterm milk, and 18 were present at higher levels in term milk.     

Identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.     

Identification of leptomeningeal metastasis-related proteins in cerebrospinal fluid of patients with breast cancer by a combination of MALDI-TOF, MALDI-FTICR and nanoLC-FTICR MS

Leptomeningeal metastasis (LM) is a devastating complication occurring in 5% of breast cancer patients. However, the current 'gold standard' of diagnosis, namely microscopic examination of the cerebrospinal fluid (CSF), is false-negative in 25% of patients at the first lumbar puncture. In a previous study, we analyzed a set of 151 CSF samples (tryptic digests) by MALDI-TOF and detected peptide masses that were differentially expressed in breast cancer patients with LM. In the present study, we obtain for a limited number of samples exact masses for these peptides by MALDI-FTICR MS measurements. Identification of these peptides was performed by electrospray FTICR MS after separation by nano-scale LC. The database results were confirmed by targeted high mass accuracy measurements of the fragment ions in the FTICR cell. The combination of automated high-throughput MALDI-TOF measurements and analysis by FTICR MS leads to the identification of 17 peptides corresponding to 9 proteins. These include proteins that are operative in host-disease interaction, inflammation and immune defense (serotransferrin, alpha 1-antichymotrypsin, hemopexin, haptoglobin and transthyretin). Several of these proteins have been mentioned in the literature in relation to cancer. The identified proteins alpha1-antichymotrypsin and apolipoprotein E have been described in relation to Alzheimer's disease and brain cancer.     

Mass Spectrometric Identification and Quantification of Hemorphins Extracted from Human Adrenal and Pheochromocytoma Tissue

Abstract: The hemorphins are a family of recently identified opioid receptor binding peptides derived from the proteolytic processing of the β, γ, δ, and ε chains of hemoglobin. They have previously been identified at high concentration in human pituitary glands and in the CSF of patients with cerebral bleeding. Hemorphins are potent inhibitors of angiotensin converting enzyme and therefore possibly have a role to play in blood pressure regulation. We report the presence of four hemorphin peptides in extracts of normal adrenal tissue and in pheochromocytoma tumors. The hemorphins were quantified and structurally characterized using mass spectrometry. High concentrations of hemorphins were found in all samples, comparable with the levels reported in the literature for pituitary and brain tissue.     

Autoantibody profiling in multiple sclerosis reveals novel antigenic candidates

An important contribution of B cells and autoantibodies has been demonstrated in the pathogenesis of multiple sclerosis (MS), leading to interest in the use of such autoantibodies as diagnostic or prognostic biomarkers. The objective of this study was to identify novel Ab biomarkers for MS using "serological Ag selection". Using a phage display library derived from MS brain plaques, we applied serological Ag selection to identify antigenic targets specifically interacting with Abs present in the cerebrospinal fluid (CSF) of 10 relapsing-remitting MS patients. These antigenic targets were further evaluated on a large panel of CSF from 63 other MS patients, 30 patients with other inflammatory disorders, and 64 patients with noninflammatory neurological disorders. A panel of eight antigenic targets was identified that showed a 86% specificity and 45% sensitivity in discriminating MS patients and controls. Four of the antigenic targets showed exclusive reactivity (100% specificity; 23% sensitivity) in the MS group as compared with the control group. Detailed bio-informatic analyses revealed a novel Ag, SPAG16. Among the novel phage peptides identified, novel epitopes were generated from untranslated sequences and out-of-frame sequences. Of 10 relapsing-remitting patients used for serological Ag selection, Ab reactivity toward one of the eight antigenic targets was also demonstrated in serum of 38% CSF-positive patients. Autoantibody profiles against epitopes derived from MS brain tissue could serve as diagnostic markers or form the basis for the identification of a subgroup of MS patients.     

Discovery of neuropeptides in the nematode Ascaris suum by database mining and tandem mass spectrometry

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to discover peptides in extracts of the large parasitic nematode Ascaris suum. This required the assembly of a new database of known and predicted peptides. In addition to those already sequenced, peptides were either previously predicted to be processed from precursor proteins identified in an A. suum library of expressed sequence tags (ESTs) or newly predicted from a library of A. suum genome survey sequences (GSSs). The predicted MS/MS fragmentation patterns of this collection of real and putative peptides were compared with the actual fragmentation patterns found in the MS/MS spectra of peptides fractionated by MS; this enabled individual peptides to be sequenced. Many previously identified peptides were found, and 21 novel peptides were discovered. Thus, this approach is very useful, despite the fact that the available GSS database is still preliminary, having only 1× coverage.     

Analysis of the rat hypothalamus proteome by data‐independent label‐free LC‐MS/MS

Studies of neuronal, endocrine, and metabolic disorders would be facilitated by characterization of the hypothalamus proteome. Protein extracts prepared from 16 whole rat hypothalami were measured by data‐independent label‐free nano LC‐MS/MS. Peptide features were detected, aligned, and searched against a rat Swiss‐Prot database using ProteinLynx Global Server v.2.5. The final combined dataset comprised 21 455 peptides, corresponding to 622 unique proteins, each identified by a minimum of two distinct peptides. The majority of the proteins (69%) were cytosolic, and 16% were membrane proteins. Important proteins involved in neurological and synaptic function were identified including several members of the Ras‐related protein family and proteins involved in glutamate biosynthesis.     

Peptides in the cerebrospinal fluid of neuropsychiatric patients: An approach to central nervous system peptide function

This review highlights that essentially all of the recently discovered putative central nervous system (CNS) peptides and other peptide substances are measurable in human cerebrospinal fluid (CSF). Preliminary evidence also suggests that peptides in CSF may have an active regulatory role in relation to CNS function and behavior. Even if this is not the case, CSF peptides may prove to be a useful indirect marker of CNS peptide function and metabolism. Alterations in peptides have been reported in neurological and psychiatric illness, pain symptoms and their treatment, symptoms such as anxiety, and following treatment with CNS active drugs such as carbamazepine. CSF methodologies provide a strategy for the study of the interaction of classical neurotransmitters and peptide substances and their relationship to neural function and behavior in man. Assessment of peptides in CSF may supplement post mortem studies of peptide levels and receptor distribution and help lead to new diagnostic and treatment approaches in neuropsychiatric disorders.     

Identification of highly acidic peptides from processing of the skin prepropeptides of Xenopus laevis

The skin secretion of the frog Xenopus laevis has been fractionated by reverse-phase HPLC and the most polar components studied by fast-atom-bombardment mass spectrometry (FAB/MS). Esterification of the hydrophilic peptides with methanol and ethanol was employed to improve the sensitivity of the technique. A number of small, highly acidic peptides have been identified, and alcoholysis of the peptide bonds within a number of these permitted their sequencing by FAB/MS. The sequences confirmed that they originate from acidic spacer regions found in the precursors to peptide hormones, such as caerulein, which have already been found in the secretion. In addition, acidic peptides derived from the spaces of the precursor to the antimicrobial peptides, PGS (or the magainins) have been isolated. The release of these from the preprotein cannot be fully accounted for by documented processing mechanisms, suggesting that a novel type of cleavage site has been identified.     

Phosphotyrosine profiling of human cerebrospinal fluid

Background

Cerebrospinal fluid (CSF) is an important source of potential biomarkers that affect the brain. Biomarkers for neurodegenerative disorders are needed to assist in diagnosis, monitoring disease progression and evaluating efficacy of therapies. Recent studies have demonstrated the involvement of tyrosine kinases in neuronal cell death. Thus, neurodegeneration in the brain is related to altered tyrosine phosphorylation of proteins in the brain and identification of abnormally phosphorylated tyrosine peptides in CSF has the potential to ascertain candidate biomarkers for neurodegenerative disorders.

Methods

In this study, we used an antibody-based tyrosine phosphopeptide enrichment method coupled with high resolution Orbitrap Fusion Tribrid Lumos Fourier transform mass spectrometer to catalog tyrosine phosphorylated peptides from cerebrospinal fluid. The subset of identified tyrosine phosphorylated peptides was also validated using parallel reaction monitoring (PRM)-based targeted approach.

Results

To date, there are no published studies on global profiling of phosphotyrosine modifications of CSF proteins. We carried out phosphotyrosine profiling of CSF using an anti-phosphotyrosine antibody-based enrichment and analysis using high resolution Orbitrap Fusion Lumos mass spectrometer. We identified 111 phosphotyrosine peptides mapping to 66 proteins, which included 24 proteins which have not been identified in CSF previously. We then validated a set of 5 tyrosine phosphorylated peptides in an independent set of CSF samples from cognitively normal subjects, using a PRM-based targeted approach.

Conclusions

The findings from this deep phosphotyrosine profiling of CSF samples have the potential to identify novel disease-related phosphotyrosine-containing peptides in CSF.

     

Use of peptide analogue diversity library beads for increased depth of proteomic analysis: application to cerebrospinal fluid

Biological samples can contain proteins with concentrations that span more than 10 orders of magnitude. Given the limited dynamic range of analysis methods, observation of proteins present at the lower concentrations requires depletion of high-abundance proteins, or other means of reducing the dynamic range of concentrations. Hexapeptide diversity library beads have been used to bind proteins in a complex sample up to a given saturation limit, effectively truncating the maximum concentration of proteins at a desired level. To avoid the potential problem of susceptibility of the hexapeptides to cleavage by proteases in the sample and/or bacterial degradation, peptide analogues that exhibit similar binding characteristics to peptides can be used in place of peptides. We report here the use of hexameric peptoid diversity library beads to reduce the dynamic range of protein concentrations in human cerebrospinal fluid (CSF). Using this method in conjunction with 2D LC/MS/MS analyses, we identified 200 unique proteins, about twice the number identified in untreated CSF.     

Identification of isoflavone glycosides in Pueraria lobata cultures by tandem mass spectrometry

Isoflavones in the methanolic extracts of kudzu (Pueraria lobata) callus, suspension and root cultures were compared in order to develop an experimental system in which puerarin (daidzein 8-C-glucoside) and other isoflavones could be synthesised in vitro. Quantitative variation of puerarin and other known isoflavones was estimated in kudzu culture extracts using HPLC-UV. The highest and lowest amounts of puerarin (14.56 and 0.33 mg/g) were present in in vitro root cultures and leaf tissue-derived callus cultures, respectively. A total of 48 isoflavone metabolites were detected in extracts of kudzu root cultures by HPLC-MS/MS, and the structures of 33 of them were tentatively assigned. Amongst these, 12 isoflavone C-glycosides were identified. Hydroxyderivatives of puerarin in several isomeric forms were detected, some of which have not been previously reported in kudzu root. The molecular weights, interpretation of characteristic fragment ions obtained from HPLC-MS/MS and comparison with reported data allowed the putative identification of the isoflavone metabolites.     

Four-dimensional proteomics analysis of human cerebrospinal fluid with trapped ion mobility spectrometry using PASEF

A quadrupole time-of-flight mass spectrometer coupled with a trapped ion mobility spectrometry (timsTOF) operated in parallel accumulation-serial fragmentation (PASEF) mode has recently emerged as a platform capable of providing four-dimensional (4D) features comprising of elution time, collision cross section (CCS), mass-to-charge ratio, and intensity of peptides. The PASEF mode provides ∼100% ion sampling efficiency both in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes without sacrificing sensitivity. In addition, targeted measurements using PASEF integrated parallel reaction monitoring (PRM) mode have also been described. However, only limited number of studies have used timsTOF for analysis of clinical samples. Although Orbitrap mass spectrometers have been used for biomarker discovery from cerebrospinal fluid (CSF) in a variety of neurological diseases, these Orbitrap-derived datasets cannot readily be applied for driving experiments on timsTOF mass spectrometers. We generated a catalog of peptides and proteins in human CSF in DDA mode on a timsTOF mass spectrometer and used these data to build a spectral library. This strategy allowed us to use elution times and ion mobility values from the spectral library to design PRM experiments for quantifying previously discovered biomarkers from CSF samples in Alzheimer's disease. When the same samples were analyzed using a DIA approach combined with a spectral library search, a higher number of proteins were identified than in a library-free approach. Overall, we have established a spectral library of CSF as a resource and demonstrated its utility for PRM and DIA studies, which should facilitate studies of neurological disorders.     

Integrated analysis of the cerebrospinal fluid peptidome and proteome

Cerebrospinal fluid (CSF) is the only body fluid in direct contact with the brain and thus is a potential source of biomarkers. Furthermore, CSF serves as a medium of endocrine signaling and contains a multitude of regulatory peptides. A combined study of the peptidome and proteome of CSF or any other body fluid has not been reported previously. We report confident identification in CSF of 563 peptide products derived from 91 precursor proteins as well as a high confidence CSF proteome of 798 proteins. For the CSF peptidome, we use high accuracy mass spectrometry (MS) for MS and MS/MS modes, allowing unambiguous identification of neuropeptides. Combination of the peptidome and proteome data suggests that enzymatic processing of membrane proteins causes release of their extracellular parts into CSF. The CSF proteome has only partial overlap with the plasma proteome, thus it is produced locally rather than deriving from plasma. Our work offers insights into CSF composition and origin.     

Volatile components from the mandibular glands of the turtle ants, Cephalotes alfaroi and Cephalotes cristatus

GC–MS analysis of whole head extracts from the turtle ants, Cephalotes alfaroi and Cephalotes cristatus, showed that 4-heptanone and 4-heptanol were the major volatile components in the mandibular glands. 4-Heptanone and 4-heptanol have rarely been identified in mandibular gland secretions from other ant genera. Thus, these compounds may be chemotaxonomic markers for the genus Cephalotes, since they have been identified in the mandibular glands from all members of this genus that have been investigated to date. Minor components identified in the whole head extracts of these ants were 3-methyl-1-butanol, 3-heptanone, 3-hexanol, 2- and 3-methylbutanoic acids, 2-methyl-4-heptanone, 2-phenylethanol and phenol. To our knowledge, this is the first time that 2-methyl-4-heptanone and phenol have been reported in the mandibular gland secretion from any Formicid.     

Age‐related cleavages of crystallins in human lens cortical fiber cells generate a plethora of endogenous peptides and high molecular weight complexes

Low molecular weight peptides derived from the breakdown of crystallins have been reported in adult human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilization of crystallins. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of four human lenses representing young, middle and old‐age human lenses. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C‐terminus with significantly higher frequency compared to other residues, suggesting that trypsin‐like proteolysis may be active in the lens cortical fiber cells. Selected reaction monitoring analysis of an endogenous αA‐crystallin peptide (αA_57‐65) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA_57‐65 formation escalated significantly. Using 2D gel electrophoresis/nanoLC‐ESI‐MS/MS, 12 protein complexes of 40–150 kDa consisting of multiple crystallin components were characterized from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross‐linking, with these complexes potentially acting to stabilize degraded crystallins by sequestration into water soluble complexes. Proteins 2015; 83:1878–1886. © 2015 Wiley Periodicals, Inc.     

Quantitative proteomic analysis of LPS-induced differential immune response associated with TLR4 Polymorphisms by multiplex amino acid coded mass tagging

Polymorphisms at toll-like receptor 4 (TLR4) gene have been found to be associated with immune disorders. A murine macrophage cell line GG2EE derived from C3H/HeJ mice with a polymorphism site at TLR4 is hyposensitive to lipopolysaccharide (LPS). To study the molecular base of diverse TLR4-mediated immune responses, the proteomic changes in both TLR4-deficient and wild-type cell lines in response to the same LPS challenge were quantitatively compared by using multiplex amino acid coded mass tagging (AACT)/SILAC-assisted MS. This strategy allows encoding of two distinct cell populations with different stable isotope-tagged lysine residues as the "in-spectra" quantitative markers. In MS analysis of tryptic peptides derived from the equally mixed three cell populations, the lysine-containing peptides originated from two LPS-stimulated cell populations can be clearly distinguished by their different mass shifts from the unstimulated and unlabeled counterpart. The LPS-induced differential protein expression in TLR4-deficient and wild-type proteomes were obtained by comparing the intensities of isotopically encoded peptides. Among the more than 900 proteins identified, 35 were found to be deregulated at different levels in these two cell lines stimulated by LPS. This multiplex mass-tagging methodology can be readily extended to other comparative proteomic quantitation of different cell populations.     

Application of matrix solid-phase dispersion methodology to the extraction of endogenous peptides from porcine hypothalamus samples for MS and LC-MS analysis

In this study, we investigated a novel application of matrix solid-phase dispersion (MSPD) methodology for the extraction of endogenous peptides from porcine hypothalamus tissue samples. Several experimental factors of the MSPD procedure were examined. Finally, silica-based octadecyl was chosen as dispersing material and blended with 0.25 g porcine hypothalamus at a ratio of 5, and 10 mL of 60% acetonitrile with 0.2% formic acid in water was chosen as the extraction and elution solvent. This MSPD extraction method was compared to the classic acid extraction method. More peaks were observed in the MSPD extracts (74±5) by MALDI-TOF MS than in acid extracts (34±5). Moreover, 14 potential endogenous peptides were identified in the MSPD extracts after nanoLC-MS/MS analysis, while only 2 endogenous peptides in the acid extracts. These results indicated that MSPD could be employed as a simple and efficient method for the extraction of endogenous peptides from tissues.     

CSF N-Glycan Profiles to Investigate Biomarkers in Brain Developmental Disorders: Application to Leukodystrophies Related to eIF2B Mutations

Background

Primary or secondary abnormalities of glycosylation have been reported in various brain diseases. Decreased asialotransferrin to sialotransferrin ratio in cerebrospinal fluid (CSF) is a diagnostic marker of leukodystrophies related to mutations of genes encoding translation initiation factor, EIF2B. We investigated the CSF glycome of eIF2B-mutated patients and age-matched normal individuals in order to further characterize the glycosylation defect for possible use as a biomarker.

Methodology/Principal Findings

We conducted a differential N-glycan analysis using MALDI-TOF/MS of permethylated N-glycans in CSF and plasma of controls and eIF2B-mutated patients. We found in control CSF that tri-antennary/bisecting and high mannose structures were highly represented in samples obtained between 1 to 5 years of age, whereas fucosylated, sialylated structures were predominant at later age. In CSF, but not in plasma, of eIF2B-mutated patient samples, we found increased relative intensity of bi-antennary structures and decreased tri-antennary/bisecting structures in N-glycan profiles. Four of these structures appeared to be biomarker candidates of glycomic profiles of eIF2B-related disorders.

Conclusion

Our results suggest a dynamic development of normal CSF N-glycan profiles from high mannose type structures to complex sialylated structures that could be correlated with postnatal brain maturation. CSF N-glycome analysis shows relevant quantitative changes associated with eIF2B related disorders. This approach could be applied to other neurological disorders involving developmental gliogenesis/synaptogenesis abnormalities.