Purification and characterization of recombinant malate synthase enzymes from Streptomyces coelicolor A3(2) and S. clavuligerus NRRL3585

Malate synthases (MS) from Streptomyces coelicolor A3(2) and S. clavuligerus NRRL3585 were cloned by polymerase chain reaction into a glutathione S-transferase (GST) fusion expression vector and heterologously expressed in Escherichia coli. The fusion GST–MS construct improved the soluble expression of MS by approximately 10-fold compared to the soluble expression of nonfusion MS. With the significant improvement in levels of soluble MS, purification and subsequent cleavage of recombinant MS from GST were facilitated in this study. Using purified enzymes, optimized parameters, which achieved maximal specific activity, were established in the enzymatic assay for streptomycete MS. The average purified specific activities of S. coelicolor and S. clavuligerus MS were 26199 and 11821 nmol/mg min, respectively. Furthermore, enzymatic analysis revealed that the two streptomycete MS displayed a similar K _m value for acetyl-CoA, but S. coelicolor MS had a K _m value for glyoxylate that is approximately sixfold higher than S. clavuligerus MS. Journal of Industrial Microbiology & Biotechnology (2002) 28, 239–243 DOI: 10.1038/sj/jim/7000240 Received 09 July 2001/ Accepted in revised form 27 December 2001     

Protective effect of Zizyphus lotus jujube fruits against cypermethrin-induced oxidative stress and neurotoxicity in mice

Context: Cypermethrin (CYP) is a synthetic pyrethroid insecticide used worldwide in agriculture, home pest control. The toxicity of CYP is well studied in many organisms.

Objective: The aim of present study was to investigate the protective effect of Zizyphus lotus (Zizyp) fruit against neurotoxicity and oxidative stress induced by CYP in mice.

Materials and methods: Mice were divided into four groups of six each: groups I and II were used as control and CYP control (20?mg/kg body weight). While, groups III was orally treated with Zizyphus lotus fruit (5?g/kg body weight) plus CYP (20?mg/kg body weight) for 18?days. Furthermore, HPLC–ESI–MS–MS (Q-Tof) and GC–MS were used to identify the compounds fraction.

Results: Antioxidant enzyme catalase (CAT), neurotoxicity enzyme acetylcholinesterase (AChE) activities and hydrogen peroxide (H₂O₂)_, malondialdehyde (MDA) levels were determined in the liver, kidney and heart. CYP caused decreased CAT activity, inhibition of AChE activity and increased the levels of H₂O₂ and MDA in heart, liver and kidney.

Conclusion: Our results indicate that Zizyp fruit is markedly effective in protecting mice against CYP-induced biochemical changes. This protection may be due to its antioxidant property and scavenging ability against active free radicals.     

Structure elucidation of ostreocin-A and ostreocin-E1, novel palytoxin analogs produced by the dinoflagellate Ostreopsis siamensis,using LC/Q-TOF MS

Palytoxin analogs are marine toxins with large complex polyol structures. A benthic dinoflagellate Ostreopsis siamensis produces more than ten palytoxins (ostreocins, OSTs). The limited sample availability of minor OSTs restricts the definition of their chemical structures. The present investigation characterizes structures of two minor OSTs, i.e., ostreocin-A (OSTA) and ostreocin-E1 (OSTE1), using ostreocin-D (OSTD) as a reference compound, by liquid chromatography/quadrupole-time-of-flight mass spectrometry. The molecular formulas of OSTA and OSTE1 were C₁₂₇H₂₁₉N₃O₅₄ and C₁₂₇H₂₁₇N₃O₅₂, respectively. Compared to OSTD, OSTA has an extra oxygen atom whereas OSTE1 lacks one oxygen atom and two hydrogen atoms. The MS/MS experiments (precursor ions: [M + H]⁺ and [M-H]^?) suggested a hydroxyl substitution at C82 in OSTA and alteration(s) between C53 and C100 in OSTE1. Further analysis of structural details in OSTE1 was performed through a pseudo-MS³ experiment (precursor ion: m/z 1432.748). Accordingly, the planar structures of OSTA and OSTE1 were assigned to 42,82-dihydroxy-3,26-didemethyl-19,44-dideoxypalytoxin and 42-hydroxy-3,26-didemethyl-19,44,73-trideoxypalytoxin-72-ene, respectively.

Abbreviations:CID: collision induced dissociation; HR-LC/MS/MS: high-resolution liquid chromatography/tandem mass spectrometry; LC/ESI/Q-TOF MS: liquid chromatography/quadrupole time-of-flight mass spectrometry equipped with an electrospray ionization source; NMR: nuclear magnetic resonance; OSTs: ostreocins; OSTA: ostreocin-A; OSTB: ostreocin-B; OSTD: ostreocin-D; OSTE1: ostreocin-E1; OVTX-a: ovatoxin-a; OVTXs: ovatoxins; PLTX: palytoxin     

LC/MS/MS method for analysis of E₂ series prostaglandins and isoprostanes

15-series prostaglandins (PGE₂s) and isoprostanes (isoPGE₂s) are robust biomarkers of oxidative stress, possess potent biological activity, and may be derived through cyclooxygenase or free radical pathways. Thus, their quantification is critical in understanding many biological processes where PG, isoPG, or oxidative stress are involved. LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE₂/D₂ and isoPGE₂ without derivatization and multiple HPLC separations. The developed LC/MS/MS method allows for the major brain PGE₂/PGD₂/isoPGE₂ such as PGE₂, entPGE₂, 8-isoPGE₂, 11β-PGE₂, PGD₂, and 15(R)-PGD₂ to be separated and quantified without derivatization. The method was validated by analyzing free and esterified isoPGE₂ in mouse brains fixed with head-focused microwave irradiation before or after global ischemia. Using the developed method, we report for the first time the esterified isoPGE₂ levels in brain tissue under basal conditions and upon global ischemia and demonstrate a nonreleasable pool of esterified isoPG upon ischemia. In addition, we demonstrated that PGE₂s found esterified in the sn-2 position in phospholipids are derived from a free radical nonenzymatic pathway under basal conditions. Our method for brain PG analysis provides a high level of selectivity to detect changes in brain PG and isoPG mass under both basal and pathological conditions.     

Vascular and Hepatic Impact of Short-Term Intermittent Hypoxia in a Mouse Model of Metabolic Syndrome

BackgroundExperimental models of intermittent hypoxia (IH) have been developed during the last decade to investigate the consequences of obstructive sleep apnea. IH is usually associated with detrimental metabolic and vascular outcomes. However, paradoxical protective effects have also been described depending of IH patterns and durations applied in studies. We evaluated the impact of short-term IH on vascular and metabolic function in a diet-induced model of metabolic syndrome (MS).MethodsMice were fed either a standard diet or a high fat diet (HFD) for 8 weeks. During the final 14 days of each diet, animals were exposed to either IH (1 min cycle, FiO₂ 5% for 30s, FiO₂ 21% for 30s; 8 h/day) or intermittent air (FiO₂ 21%). Ex-vivo vascular reactivity in response to acetylcholine was assessed in aorta rings by myography. Glucose, insulin and leptin levels were assessed, as well as serum lipid profile, hepatic mitochondrial activity and tissue nitric oxide (NO) release.ResultsMice fed with HFD developed moderate markers of dysmetabolism mimicking MS, including increased epididymal fat, dyslipidemia, hepatic steatosis and endothelial dysfunction. HFD decreased mitochondrial complex I, II and IV activities and increased lactate dehydrogenase (LDH) activity in liver. IH applied to HFD mice induced a major increase in insulin and leptin levels and prevented endothelial dysfunction by restoring NO production. IH also restored mitochondrial complex I and IV activities, moderated the increase in LDH activity and liver triglyceride accumulation in HFD mice.ConclusionIn a mouse model of MS, short-term IH increases insulin and leptin levels, restores endothelial function and mitochondrial activity and limits liver lipid accumulation.     

The role of mass spectrometry in the characterization of biologic protein products

Introduction: Mass spectrometry (MS) is widely used in the characterization of biomolecules including peptide and protein therapeutics. These biotechnology products have seen rapid growth over the past few decades and continue to dominate the global pharmaceutical market. Advances in MS instrumentation and techniques have enhanced protein characterization capabilities and supported an increased development of biopharmaceutical products.

Areas covered: This review describes recent developments in MS-based biotherapeutic analysis including sequence determination, post-translational modifications (PTMs) and higher order structure (HOS) analysis along with improvements in ionization and dissociation methods. An outlook of emerging applications of MS in the lifecycle of product development such as comparability, biosimilarity and quality control practices is also presented.

Expert commentary: MS-based methods have established their utility in the analysis of new biotechnology products and their lifecycle appropriate implementation. In the future, MS will likely continue to grow as one of the leading protein identification and characterization techniques in the biopharmaceutical industry landscape.     

Proteomics analysis to compare the venom composition between Naja naja and Naja kaouthia from the same geographical location of eastern India: Correlation with pathophysiology of envenomation and immunological cross-reactivity towards commercial polyantivenom

ABSTRACT

Background : Cobra bite is frequently reported across the Indian subcontinent and is associated with a high rate of death and morbidity. In eastern India (EI) Naja naja and Naja kaouthia are reported to be the two most abundant species of cobra.

Research design and methods : The venom proteome composition of N. naja (NnV) and N. kaouthia (NkV) from Burdwan districts of EI were compared by separation of venom proteins by 1D-SDS-PAGE followed by LC-MS/MS analysis of protein bands. The potency of commercial polyantivenom (PAV) was assessed by neutralization, ELISA, immuno-blot and venom-PAV immunoaffinity chromatography studies.

Results : Proteomic analysis identified 52 and 55 proteins for NnV and NkV, respectively, when searched against the Elapidae database. A small quantitative difference in venom composition between these two species of cobra was observed. PAVs exhibited poor cross-reactivity against low molecular mass toxins (<20 kDa) of both cobra venoms, which was substantiated by a meager neutralization of their phospholipase A₂ activity. Phospholipase A₂ and 3FTx, the two major classes of nonenzymatic and enzymatic proteins, respectively, were partially recognized by PAVs.

Conclusions : Efforts must be made to improve immunization protocols and supplement existing antivenoms with antibodies raised against the major toxins of these venoms.     

Molecular-networking-guided discovery of species-specific markers for discriminating five medicinal Paris herbs

BackgroundParidis Rhizoma (PR) is a famous traditional herbal medicine. Apart from two officially recorded species, viz. Paris polyphylla Smith var. yunnanensis (Franch.) Hand. - Mazz. (PPY) and P. polyphylla Smith var. chinensis (Franch.) Hara (PPC), there are still many other species used as folk medicine. It is necessary to understand the metabolic differences among Paris species.PurposeTo establish a strategy that can discover species-specific steroidal saponin markers to distinguish closely-related Paris herbs for quality and safety control.MethodsA new strategy of molecular-networking-guided discovery of species-specific markers was proposed. Firstly, the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was applied to obtain the MS and MS/MS data of all samples. Then, molecular networking (MN) was created using MS/MS data to prescreen the steroidal saponins for subsequent analysis. Next, the principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) models were established to discover potential markers. Finally, the verification, identification and distribution of chemical markers were performed.ResultsA total of 126 steroidal saponins were screened out from five species using MN. Five species were classified successfully by OPLS-DA model, and 18 species-specific markers were discovered combining the variable importance in the projection (VIP) value, P value (one-way ANOVA) and their relative abundance. These markers could predict the species of Paris herbs correctly.ConclusionThese results revealed that this new strategy could be an efficient way for chemical discrimination of medicinal herbs with close genetic relationship.     

Spectral analysis and in vitro cytotoxicity profiles of novel organotin(IV) esters of 2-maleimidopropanoic acid

Six novel triorganotin(IV) 2-maleimidopropanoato complexes: R₃SnOCOCH₃(CH)(COCH)₂, (R: Me(1), Et(2), n-Pr(3), n-Bu(4), Ph(5), Bz(6) have been synthesized. Their solid-state configuration has been determined by FT IR and ^119mSn Mössbauer spectroscopy. The tin(IV) atom is five-coordinated in all the complexes with 2-maleimidopropanoic acid behaving as a monoanionic bidentate ligand coordinating the tin(IV) atom through a chelating or bridging carboxylate group. The solution-state configuration has been elucidated by means of ¹H-, ¹³C- and ¹¹⁹Sn-NMR spectroscopy which assigned a tetrahedron. Elemental analysis and FAB MS data also supported a 1:1 metal to ligand stoichiometry. The title complexes have been screened in vitro for anti-tumour, anti-fungal, anti-leishmanial and urease inhibition activities and displayed promising results.     

Current development in non-enzymatic lipid peroxidation products,isoprostanoids and isofuranoids,in novel biological samples

Abstract

Isoprostanoids and isofuranoids are lipid mediators that can be formed from omega-3 and omega-6 polyunsaturated fatty acids (PUFAs). F₂-isoprostanes formed from arachidonic acid, especially 15-F_2t-isoprostane, are commonly measured in biological tissues for decades as the biomarker for oxidative stress and diseases. Recently, other forms of isoprostanoids derived from adrenic, eicosapentaenoic, and docosahexaenoic acids namely F₂-dihomo-isoprostanes, F₃-isoprostanes, and F₄-neuroprostanes respectively, and isofuranoids including isofurans, dihomo-isofurans, and neurofurans are reported as oxidative damage markers for different metabolisms. The most widely used samples in measuring lipid peroxidation products include but not limited to the blood and urine; other biological fluids, specialized tissues, and cells can also be determined. In this review, measurement of isoprostanoids and isofuranoids in novel biological samples by gas chromatography (GC)–mass spectrometry (MS), GC–MS/MS, liquid chromatography (LC)–MS, and LC–MS/MS will be discussed.     

Anti-inflammatory action of herbal medicine comprised of Scutellaria baicalensis and Chrysanthemum morifolium

ABSTRACT

Various mixtures were prepared depending on the mixing ratio of Scutellaria baicalensis hot water extract (SB-HW), and Chrysanthemum morifolium ethanol extract (CM-E) and their anti-inflammatory activity were compared. Among them, SB-HW (80 μg/mL)/CM-E (120 μg/mL) or SB-HW (40 μg/mL)/CM-E (160 μg/mL) significantly inhibited LPS-stimulated NO and IL-6 levels in RAW 264.7 cells. The SB-HW (80 μg/mL)/CM-E (120 μg/mL) mixture, which was determined as active mixture, significantly reduced MUC5AC secretion in PMA and LPS-induced NCI-H292 cells. The active mixture also reduced the production of PGE₂ and IL-8 in PMA-induced A549 cells. LC-MS/MS analysis showed that the active mixture was composed of high contents of flavone glycosides, such as baicalin and cynaroside. Western blot analysis indicated that the active mixture suppressed phosphorylation of ERK, JNK, and p38, associating with the inhibition of MAPK signaling. Taken together, our results suggest that the active mixture could be applied as a new anti-inflammatory herbal medicine.     

Systematic considerations for a multicomponent pharmacokinetic study of Epimedii wushanensis herba: From method establishment to pharmacokinetic marker selection

BackgroundPrenylflavonoids are major active components of Epimedii wushanensis herba (EWH). The global pharmacokinetics of prenylflavonoids are unclear, as these compounds yield multiple, often unidentified metabolites.PurposeThis study successfully elucidated the pharmacokinetic profiles of EWH extract and five EWH-derived prenylflavonoid monomers in rats.Study designThe study was a comprehensive analysis of metabolic pathways and pharmacokinetic markers.MethodsMajor plasma compounds identified after oral administration of EWH-derived prototypes or extract included: (1) prenylflavonoid prototypes, (2) deglycosylated products, and (3) glucuronide conjugates. To select appropriate EWH-derived pharmacokinetic markers, a high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was established to simultaneously monitor 14 major compounds in unhydrolyzed plasma and 10 potential pharmacokinetic markers in hydrolyzed plasma.ResultsThe pharmacokinetic profiles indicated that the glucuronide conjugates of icaritin were the principle circulating metabolites and that total icaritin accounted for ∼99% of prenylflavonoid exposure after administration of EWH-derived materials to rats. To further investigate icaritin as a prospective pharmacokinetic marker, correlation analysis was performed between total icaritin and its glucuronide conjugates, and a strong correlation (r > 0.5) was found, indicating that total icaritin content accurately reflected changes in the exposure levels of the glucuronide conjugates over time. Therefore, icaritin is a sufficient pharmacokinetic marker for evaluating dynamic prenylflavonoid exposure levels. Next, a mathematical model was developed based on the prenylflavonoid content of EWH and the exposure levels in rats, using icaritin as the pharmacokinetic marker. This model accurately predicted exposure levels in vivo, with similar predicted vs. experimental area under the curve (AUC)_0–96 h values for total icaritin (24.1 vs. 32.0 mg/L h).ConclusionIcaritin in hydrolyzed plasma can be used as a pharmacokinetic marker to reflect prenylflavonoid exposure levels, as well as the changes over time of its glucuronide conjugates.     

On the soybean type-I lipoxygenase-mediated conversion of linoleic acid into (S) 2-nonen-4-olide

(S) 2-nonen-4-olide3 of 0.5ee is obtained upon steam distillation from linoleic acid1 treated with oxygen at pH 9.6 in the presence of soybean type-I lipoxygenase; the two enantiomers of 2-nonen-4-olide obtained from [12, 13-²H₂]1 were shown by multidimensional GC/MS to contain both 70% d₂ and 30% d₁ species     

Synthesis of novel sulfonamides under mild conditions with effective inhibitory activity against the carbonic anhydrase isoforms I and II

Novel sulfonamide derivatives 6a–i, as new carbonic anhydrase inhibitors which candidate for glaucoma treatment, were synthesized from the reactions of 4-amino-N-(4-sulfamoylphenyl) benzamide 4 and sulfonyl chloride derivatives 5a–i with high yield (71–90%). The structures of these compounds were confirmed by using spectral analysis (FT-IR, ¹H NMR, ¹³C NMR, LC/MS and HRMS). The inhibition effects of 6a–i on the hydratase and esterase activities of human carbonic anhydrase isoenzymes, hCA I and II, which were purified from human erythrocytes with Sepharose®4B-l-tyrosine-p-aminobenzene sulfonamide affinity chromatography, were studied as in vitro, and IC₅₀ and K_i values were determined. The results show that newly synthesized compounds have quite powerful inhibitory properties.     

Oligonucleotides Incorporating 7-(Aminoalkyn-1-yl)-7-deaza-2′-deoxyguanosines: Duplex Stability and Phosphodiester Hydrolysis by Exonucleases

Abstract

Self-complementary {[5′-d(G-C)₄]₂} and non-selfcomplementary oligonucleotides [5′-d(TAG GTC AAT ACT) ? 3′-d(ATC CAG TTA TGA)] containing 7-(ω-aminoalkyn-1-yl)-7-deaza-2′-deoxyguanosines (1a–c) (1) and 7-deaza-2′-deoxyguanosine instead of dG were studied regarding their thermal stability as well as their phosphodiester hydrolysis by either 3′ → 5′- or 5′ → 3′ – phosphodi esterase studied by MALDI-TOF MS.     

A new quantitative LC tandem mass spectrometry assay for serum 25-hydroxy vitamin D

BackgroundThe accurate measurement of 25-hydoxy vitamin D (25OH-D) in serum has been a challenge for many years. We developed a liquid chromatography tandem mass spectrometry (LC Tandem MS) assay for the quantitative determination of 25OH-D₂ and 25OH-D₃ in serum. The new method was compared with two widely used commercially available immunoassays.MethodsSample preparation involved protein precipitation with acetonitrile containing deuterated forms of the target species as internal standards. An API 5000 mass spectrometer coupled with a photoionization source was used for quantitation. The performance of the new LC Tandem MS assay was compared with a radioimmunoassay (RIA, Diasorin) and a chemiluminescence immunoassay (ECLIA, Roche Diagnostics), analysing serum obtained from 152 individuals.ResultsUsing 100 μl of serum, the LC Tandem MS assay had a limit of quantitation of 1.3 nmol/L for both 25OH-D₂ and 25OH-D₃ with a linear response between 1.3 and 625 nmol/L and accuracy of between 95 and 124%. Intra- and inter-assay precision were ≤7% and ≤4%, respectively. Measurement of 25OH-D levels in 152 serum samples gave run averages of 71, 56 and 62 nmol/L for LC Tandem MS, ECLIA and RIA, respectively. Correlations between the various methods were: LC Tandem MS vs. RIA: r = 0.931; LC Tandem MS vs. ECLIA: r = 0.784; RIA vs. ECLIA: r = 0.787. The LC Tandem MS method had a positive proportional bias of 26% over the RIA, whereas the ECLIA showed variable differences.ConclusionThe new LC Tandem MS assay is accurate and precise at physiologically relevant 25OH-D concentrations, and compares favourably with the RIA. In contrast, the ECLIA shows variable bias with the other assays tested.     

Design,synthesis and molecular docking of novel diarylcyclohexenone and diarylindazole derivatives as tubulin polymerization inhibitors

New target compounds were designed as inhibitors of tubulin polymerization relying on using two types of ring B models (cyclohexenone and indazole) to replace the central ring in colchicine. Different functional groups (R¹) were attached to manipulate their physicochemical properties and/or their biological activity. The designed compounds were assessed for their antitumor activity on HCT-116 and MCF-7 cancer cell lines. Compounds 4b, 5e and 5f exhibited comparable or higher potency than colchicine against colon HCT-116 and MCF-7 tumor cells. The mechanism of the antitumor activity was investigated through evaluating the tubulin inhibition potential of the active compounds. Compounds 4b, 5e and 5f showed percentage inhibition of tubulin in both cell line homogenates ranging from 79.72% to 89.31%. Cell cycle analysis of compounds 4b, 5e and 5f revealed cell cycle arrest at G₂/M phase. Molecular docking revealed the binding mode of these new compounds into the colchicine binding site of tubulin.     

Herb-drug interaction between Styrax and warfarin: Molecular basis and mechanism

BackgroundStyrax, one of the most famous folk medicines, has been frequently used for the treatment of cardiovascular diseases and skin problems in Asia and Africa. It is unclear whether Styrax or Styrax-related herbal medicines may trigger clinically relevant herb-drug interactions.PurposeThis study was carried out to investigate the inhibitory effects of Styrax on human cytochrome P450 enzymes (CYPs) and to clarify whether this herb may modulate the pharmacokinetic behavior of the CYP-substrate drug warfarin when co-administered.Study DesignThe inhibitory effects of Styrax on CYPs were assayed in human liver microsomes (HLM), while the pharmacokinetic interactions between Styrax and warfarin were investigated in rats. The bioactive constituents in Styrax with strong CYP3A inhibitory activity were identified and their inhibitory mechanisms were carefully investigated.MethodsThe inhibitory effects of Styrax on human CYPs were assayed in vitro, while the pharmacokinetic interactions between Styrax and warfarin were studied in rats. Fingerprinting analysis of Styrax coupled with LC-TOF-MS/MS profiling and CYP inhibition assays were used to identify the constituents with strong CYP3A inhibitory activity. The inhibitory mechanism of oleanonic acid (the most potent CYP3A inhibitor occurring in Styrax) against CYP3A4 was investigated by a panel of inhibition kinetics analyses and in silico analysis.ResultsIn vitro assays demonstrated that Styrax extract strongly inhibited human CYP3A and moderately inhibited six other tested human CYPs, as well as potently inhibited warfarin 10-hydroxylation in liver microsomes from both humans and rats. In vivo assays demonstrated that compared with warfarin given individually in rats, Styrax (100 mg/kg) significantly prolonged the plasma half-life of warfarin by 2.3-fold and increased the AUC_(0-inf) of warfarin by 2.7-fold when this herb was co-administrated with warfarin (2 mg/kg) in rats. Two LC fractions were found with strong CYP3A inhibitory activity and the major constituents in these fractions were characterized by LC-TOF-MS/MS. Five pentacyclic triterpenoid acids (including epibetulinic acid, betulinic acid, betulonic acid, oleanonic acid and maslinic acid) present in Styrax were potent CYP3A inhibitors, and oleanonic acid was a competitive inhibitor against CYP3A-mediated testosterone 6β-hydroxylation.ConclusionStyrax and the pentacyclic triterpenoid acids occurring in this herb strongly modulate the pharmacokinetic behavior of warfarin via inhibition of CYP3A.     

Recent advances in applying mass spectrometry and systems biology to determine brain dynamics

Introduction: Neurological disorders encompass various pathologies which disrupt normal brain physiology and function. Poor understanding of their underlying molecular mechanisms and their societal burden argues for the necessity of novel prevention strategies, early diagnostic techniques and alternative treatment options to reduce the scale of their expected increase.

Areas covered: This review scrutinizes mass spectrometry based approaches used to investigate brain dynamics in various conditions, including neurodegenerative and neuropsychiatric disorders. Different proteomics workflows for isolation/enrichment of specific cell populations or brain regions, sample processing; mass spectrometry technologies, for differential proteome quantitation, analysis of post-translational modifications and imaging approaches in the brain are critically deliberated. Future directions, including analysis of cellular sub-compartments, targeted MS platforms (selected/parallel reaction monitoring) and use of mass cytometry are also discussed.

Expert commentary: Here, we summarize and evaluate current mass spectrometry based approaches for determining brain dynamics in health and diseases states, with a focus on neurological disorders. Furthermore, we provide insight on current trends and new MS technologies with potential to improve this analysis.     

Affinity enrichment for mass spectrometry: improving the yield of low abundance biomarkers

Introduction: Mass spectrometry (MS) is the premier tool for discovering novel disease-associated protein biomarkers. Unfortunately, when applied to complex body fluid samples, MS has poor sensitivity for the detection of low abundance biomarkers (?10 ng/mL), derived directly from the diseased tissue cells or pathogens.

Areas covered: Herein we discuss the strengths and drawbacks of technologies used to concentrate low abundance analytes in body fluids, with the aim to improve the effective sensitivity for MS discovery. Solvent removal by dry-down or dialysis, and immune-depletion of high abundance serum or plasma proteins, is shown to have disadvantages compared to positive selection of the candidate biomarkers by affinity enrichment. A theoretical analysis of affinity enrichment reveals that the yield for low abundance biomarkers is a direct function of the binding affinity (Association/Dissociation rates) used for biomarker capture. In addition, a high affinity capture pre processing step can effectively dissociate the candidate biomarker from partitioning with high abundance proteins such as albumin.

Expert commentary: Properly designed high affinity capture materials can enrich the yield of low abundance (0.1–10 picograms/mL) candidate biomarkers for MS detection. Affinity capture and concentration, as an upfront step in sample preparation for MS, combined with MS advances in software and hardware that improve the resolution of the chromatographic separation can yield a transformative new class of low abundance biomarkers predicting disease risk or disease latency.