Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data

Liquid chromatography–mass spectrometry (LC–MS) is a commonly used analytical platform for non-targeted metabolite profiling experiments. Although data acquisition, processing and statistical analyses are almost routine in such experiments, further annotation and subsequent identification of chemical compounds are not. For identification, tandem mass spectra provide valuable information towards the structure of chemical compounds. These are typically acquired online, in data-dependent mode, or offline, using handcrafted acquisition methods and manually extracted from raw data. Here, we present several methods to fast-track and improve both the acquisition and processing of LC–MS/MS data. Our nearly online (nearline) data-dependent tandem MS strategy creates a minimal set of LC–MS/MS acquisition methods for relevant features revealed by a preceding non-targeted profiling experiment. Using different filtering criteria, such as intensity or ion type, the acquisition of irrelevant spectra is minimized. Afterwards, LC–MS/MS raw data are processed with feature detection and grouping algorithms. The extracted tandem mass spectra can be used for both library search and de-novo identification methods. The algorithms are implemented in the R package MetShot and support the export to Bruker, Agilent or Waters QTOF instruments and the vendor-independent TraML standard. We evaluate the performance of our workflow on a Bruker micrOTOF-Q by comparison of automatically acquired and extracted tandem mass spectra obtained from a mixture of natural product standards against manually extracted reference spectra. Using Arabidopsis thaliana wild-type and biosynthetic gene knockout plants, we characterize the metabolic products of a biosynthetic pathway and demonstrate the integration of our approach into a typical non-targeted metabolite profiling workflow.

     

Expression of insulin-like growth factor-II,matrix metalloproteinases,and their tissue inhibitors as predictive markers in the peripheral blood of HCC patients

Background/aim: Elevated relative expression of insulin-like growth factor-II (IGF-II) was observed in hepatocellular carcinoma (HCC) liver tissues with a role in neovascularization and associated with poor prognosis. IGF-II is influenced by the proteolytic cleavage of IGF-binding protein 3 and by matrix metalloproteinases (MMP), which are further regulated by their tissue inhibitors tissue inhibitor of metalloprotienase-1 (TIMP-1). Our aim is to study new molecular markers for HCC.

Patients/methods: RNA was extracted from the peripheral blood for evaluating the relative expression of IGF-II, MMP-9, and TIMP-1 in correlation with clinical staging of 39 HCC patients and 15 healthy controls using TaqMan real-time PCR.

Results: The relative expression of IGF-II and MMP-9 mRNA were significantly elevated in HCC patients compared with healthy controls; P-value <0.0001 for both. There was a significant correlation between MMP-9 and different HCC stages. On the other hand, TIMP-1 was significantly down-regulated in HCC patients; P?=?0.0003 with the elevation of the IGF-II/TIMP-1 ratio. Significant correlation between TIMP-1 and HCC Stage III and Stage IV was found; P-value?=?0.0138.

Conclusion: These results highlight the importance of profiling the expression of IGF-II, MMP-9, and TIMP-1 in the peripheral blood as prognostic molecular biomarkers in HCC.     

The diagnostic value of serum DSA-TRF in hepatocellular carcinoma

TRF is a glycoprotein mainly secreted by hepatocytes, The aim of this study was to explore the diagnostic value of aberrant glycosylated serum transferrin (TRF) especially containing multi-antennary glycans in hepatocellular carcinoma (HCC).A total of 581 subjects including HCC patients, liver cirrhosis (LC) patients, chronic hepatitis (CHB) patients and healthy controls (HC) were recruited. All the subjects were randomly assigned to training group (n?=?411) and validation group (n?=?170). We firstly analyzed the serum protein N-glycome profiling of HCC, LC, and HC by DNA sequencer–assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) technology. We established a lectin-antibody sandwich ELISA (Lectin-ELISA) method to detect multi-antennary glycans-contained TRF (DSA-TRF) in serum, in which Datura stramonium Agglutinin (DSA) was used for specific recognition. Levels of serum DSA-TRF and TRF were analyzed respectively. The diagnostic efficacies of DSA-TRF and TRF of differentiating HCC patients from CHB, LC patients and HC within the training group were evaluated using receiver operating characteristic (ROC) curve and tested in the validation group.The result found that in training group, serum TRF and DSA-TRF levels differed significantly between HCC (1.86?±?0.50, g/L, 0.285?±?0.06), CHB?+?LC (2.39?±?0.74, g/L, 0.189?±?0.07) and HC (1.92?±?0.69, g/L, 0.249?±?0.09) (HCC vs. CHB?+?LC, P?<?0.001; HCC vs. HC, P?<?0.001; CHB?+?LC vs. HC, P?<?0.001). The area under the ROC curve (AUC) of DSA-TRF was significantly superior to AFP (0.880, 95%CI: 0.834–0.925 vs. 0.776, 95%CI: 0.725–0.827, P?=?0.003) in differentiating HCC from CHB?+?LC. The AUC of diagnostic model GlycoTRF1 (0.981, 95%CI: 0.969–0.993) was higher than DSA-TRF and AFP alone (P<0.001) in differentiating HCC from CHB?+?LC, which was verified in validation group.The results indicated that the serum DSA-TRF might serve as a potential glycan biomarker for distinguishing HCC from CHB and LC.

     

Nanoliposomes protect against AL amyloid light chain protein-induced endothelial injury

Context: A newly-recognized pathogenic mechanism underlying light chain amyloidosis (AL) involves endothelial dysfunction and cell injury caused by misfolded light chain proteins (LC). Nanoliposomes (NL) are artificial phospholipid vesicles that could attach to misfolded proteins and reduce tissue injury.

Objective: To test whether co-treatment with NL reduces LC-induced endothelial dysfunction and cell death.

Methods: Abdominal subcutaneous adipose arterioles from 14 non-AL subjects were cannulated; dilator response to acetylcholine and papaverine were measured at baseline and following 1-hour exposure to LC (20?µg/mL, 2 purified from AL subjects’ urine, 1 from human recombinant LC [AL-09])?±?NL (phosphatidylcholine/cholesterol/phosphatidic acid 70/25/5 molar ratio) or NL alone. Human aortic artery endothelial cells (HAEC) were exposed to Oregon Green-labeled LC?±?NL for 24 hours and intracellular LC and apoptosis (Hoechst stain) were measured. Circular dichroism spectroscopy was performed on AL-09 LC?±?NL to follow changes in secondary structure and protein thermal stability.

Results: LC caused impaired dilation to acetylcholine that was restored by NL (control – 94.0?±?1.8%, LC – 65.0?±?7.1%, LC?+?NL – 95.3?±?1.8%, p?≤?0.001 LC versus control or LC?+?NL). NL protection was inhibited by L-NG-nitroarginine methyl ester. NL increased the beta sheet structure of LC, reduced endothelial cell internalization of LC and protected against LC-induced endothelial cell death.

Conclusions: LC induced human adipose arteriole endothelial dysfunction and endothelial cell death, which were reversed by co-treatment with NL. This protection may partly be due to enhancing LC protein structure and reducing LC internalization. Nanoliposomes represent a promising new class of agents to ameliorate tissue injury from protein misfolding diseases such as AL.     

Phospholipids profiling and outcome of peritoneal dialysis patients

Abstract

Objectives: To investigate phospholipids (PLs) biomarkers in predicting outcome of patients undergoing peritoneal dialysis (PD).

Materials and methods: Twenty PD patients were followed using baseline plasma PLs with an improved online two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry system.

Results: Significant differences were observed in eight PL species with sphingomyelin (SM) and glycerophosphocholine between technical survival (n=15) and failure patients (n?=?5). Cox regression showed SM 21:0 (adjusted HR 13.7, 95% CI 2.42–77.88, p?=?0.003) was independently associated with patients technical failure.

Conclusions: PD failure patients had different plasma PLs profiling as compared with survival patients. Elevated plasma SM 21:0 level may potentially serve as a biomarker of PD patients at risk for adverse outcomes.     

Identification of novel serum peptides biomarkers for female breast cancer patients in Western China

This study aimed to identify novel serum peptides biomarkers for female breast cancer (BC) patients. We analyzed the serum proteomic profiling of 247 serum samples from 96 BC patients, 48 additional paired pre‐ and postoperative BC patients, 39 fibroadenoma patients as benign disease controls, and 64 healthy controls, using magnetic‐bead‐based separation followed by MALDI‐TOF MS. ClinProTools software identified 78 m/z peaks that differed among all analyzed groups, ten peaks were significantly different (P < 0.0001), with Peaks 1–6 upregulated and Peaks 7–10 downregulated in BC. Moreover, three peaks of ten (Peak 1, m/z: 2660.11; Peak 2, m/z: 1061.09; Peak 10, m/z: 1041.25) showed a tendency to return to healthy control values after surgery. And these three peptide biomarkers were identified as FGA605‐629, ITIH4 347–356, and APOA2 43–52. Methods used in this study could generate serum peptidome profiles of BC, and provide a new approach to identify potential biomarkers for diagnosis as well as prognosis of this malignancy.     

Proteomic detection of cancer in asbestosis patients using SELDI-TOF discovered serum protein biomarkers

Objectives: To identify biomarkers for cancer in asbestosis patients.

Methods: SELDI-TOF and CART were used to identify serum biomarker profiles in 35 asbestosis patients who subsequently developed cancer and 35 did not develop cancer.

Results: Three polypeptide peaks (5707.01, 6598.10, and 20,780.70?Da) could predict the development of cancer with 87% sensitivity and 70% specificity. The first two peaks were identified as KIF18A and KIF5A, respectively, and are part of the Kinesin Superfamily of proteins.

Conclusions: We identified two Kinesin proteins that can be potentially used as blood biomarkers to identify asbestosis patients at risk of developing lung cancer.     

Characterization of Novel Lactobacillus fermentum from Curd Samples of Indigenous Cows from Malnad Region,Karnataka, for their Aflatoxin B1 Binding and Probiotic Properties

Thirty-four isolates of Lactobacillus spp. (LAB) from 34 curd samples were evaluated for their aflatoxin B₁ (AFB₁) binding and probiotic properties. Upon characterization, four LAB isolates (LC3/a, LC4/c, LC/5a, and LM13/b) were found to be effective in removing AFB₁ from culture media with a capacity of above 75%. Staining reaction, biochemical tests, pattern of sugar utilization, and 16s rRNA gene sequence analysis revealed the identity of all the four isolates as L. fermentum. All of them could tolerate acidic pH, salt, and bile, which promise the use of these probiotic bacterial isolates for human applications. These isolates showed poor hydrophobicity and higher auto-aggregation properties. All L. fermentum isolates were found susceptible to gentamycin, chloramphenicol, cefoperazone, ampicillin, and resistant to ciprofloxacin and vancomycin. Results of hemolytic and DNase activity indicated their nonpathogenic nature. Though all L. fermentum isolates found inhibiting the growth of Salmonella ebony, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, maximum inhibition was obtained with isolate LC5/a. Kinetic studies revealed that all four bacteria required a minimum of 2 h to reach stationary phase of AFB₁ binding. AFB₁ binding ability varied from 66 to 85.2% among these four isolates. Bile (0.4%) was significant (P ≤ 0.05) in reducing the AFB₁ binding property of isolates LC3/a, LC4/c, and LM13/b, while increased AFB₁ binding ability was recorded at acidic pH (2.0). AFB₁ binding properties of isolate LC5/a were found least affected by acidic pH and bile. The findings of our study revealed the higher efficiency of L. fermentum isolate LC5/a in reducing the bioavailability of AFB1 in gut, and additionally, it improves the consumers’ health by its various probiotic characters. These beneficial characters, L. fermentum isolates, promise them to use as probiotic formulations alone or in combinations with other beneficial probiotic-bacterial isolates.

     

LCM development: focusing on the LC promoters and their organizational problem-solving

Purpose

Life cycle management (LCM) implies a specific sustainability perspective which extends environmental management along the product life cycle, with the aim of decreasing negative environmental impact throughout the product chain. Research has identified that the adoption of LCM in the industry depends upon its situational adaptation to the organizational context. Even so, little is known about the specifics of this adaptation. With this paper, our aim is to add knowledge on LCM adoption and adaptation.

Methods

A systematic analysis of empirical material on life cycle (LC) activity in six multinational corporations (MNCs) is conducted, by applying a secondary analysis of qualitative data (Heaton 2008). In order to study instances of LCM adoption and adaptation, we focus on the acts and situations of LC promoters. The identified instances are analyzed through the lens of situated problem-solving (Kuhn and Jackson 2008).

Results and discussion

Sixty-seven instances of LC promotion were identified and analyzed, resulting in the identification of eight categories of problem-situations typically encountered by LC promoters. The identified problem-situations represent different situations when the organizational appropriateness of the LC approach is at stake and to which responses tailored to the organization are put forward by a LC promoter. The results bring to the fore the ubiquity of organizational and creative problem-solving, highlighting the role of LC promoters as change agents for LCM adoption, and depict the development of LCM as an emergent practice, rather than an implementation process.

Conclusions

This paper provides a first systematic analysis of LC promoters enacting a variety of responses to organizationally challenging LC situations, thus detailing the adaptation necessary for embedding LCM in the industry. Findings show that the development of LCM to a great extent is about the promotion of a LC approach, and that LC promoters need organizational knowing, in addition to LC knowing, to make the LC approach relevant to management and business.

     

Dynamic changes in serum angiopoietin-1, angiopoietin-2, and angiopoietin-2/angiopoietin-1 ratio in acute myocardial infarction patients treated with primary percutaneous coronary intervention

Context: Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) play divergent roles in myocardial ischemia and reperfusion injury.

Objective: To investigate serum Ang-1 and Ang-2 levels in ST-segment elevation myocardial infarction (STEMI) patients treated with primary percutaneous coronary intervention (PCI).

Methods: Serum Ang-1 and Ang-2 were measured in 85 STEMI patients in the first week after PCI.

Results: Ang-1, Ang-2 and Ang-2/Ang-1 ratio (Ang-2/1) were all increased at admission, and had dynamic changes after PCI. Ang-2 and Ang-2/1 at admission and 2 h after PCI were positively correlated with peak cardiac troponin T levels.

Conclusion: The extent of myocardial damage may be linked to circulating Ang-2 and Ang-2/1.     

A comparison of MS/MS‐based,stable‐isotope‐labeled,quantitation performance on ESI‐quadrupole TOF and MALDI‐TOF/TOF mass spectrometers

The peptide‐based quantitation accuracy and precision of LC‐ESI (QSTAR Elite) and LC‐MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ‐labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC‐MALDI spectra. The average protein sequence coverages for LC‐ESI and LC‐MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ‐based expression ratios determined by ProteinPilot from the 57 467 ESI‐MS/MS and 26 085 MALDI‐MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7–6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC‐ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC‐MALDI iTRAQ ratios were rejected. Re‐analysis of an archived LC‐MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS‐based peptide quantitation performance of offline LC‐MALDI was comparable with on‐line LC‐ESI, which required threefold less time. However, offline LC‐MALDI allows the re‐analysis of archived HPLC‐separated samples.     

GC/MS and LC/MS Phytochemical Analysis of Vigna unguiculata L. Walp Pod

Vigna unguiculata (L. Walp) or Cowpea pod methanolic extracts phytochemical analysis, total phenolic content (TPC), and secondary metabolite profiling were determined using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) analysis. GC/MS analysis revealed twenty compounds in the extract, while LC/MS analysis identified twenty-four compounds. GC/MS chromatogram analysis suggested the presence of opioid α-N-Normethadol a major constituent found in methanolic extract and fatty acid esters carotenoid is found second major constituent. LC/MS chromatogram and the mass spectral analysis demonstrated the presence of flavonoids, carotenoids, and alkaloids as major phytochemicals. We investigated the antibacterial, anti-fungal, and anti-oxidant activity of pod methanolic extract. The extract was found equally effective against E. coli, S. pyogenes, and P. aeruginosa with MIC 100 μg/mL similar to the standard Ampicillin (MIC 100 μg/mL). C. albicans were found to be most susceptible to Vign unguiculata pods methanolic extract with a MIC of 250 μg/mL. The pod extract showed significant DPPH scavenging activity (IC₅₀=78.38±0.15) which suggests its antioxidant potential.     

The association of a distinct plasma proteomic profile with the cervical high-grade squamous intraepithelial lesion of Uyghur women: a 2D liquid-phase chromatography/mass spectrometry study

Objective: To identify plasma protein biomarkers of cervical high-grade squamous intraepithelial lesion (HSIL) of Uyghur women by proteomics approach.

Methods: Plasma protein samples of Uyghur women with HSIL and chronic cervicitis were analyzed with 2D HPLC followed by detection of target proteins with Linear Trap Quadrupole Mass Spectrometer (LTQ MS/MS).

Results: We detected three upregulated and one downregulated protein peaks representing protein constituents distinguishing HSIL from controls by 2D HPLC, identified 31 target proteins by LTQ MS/MS. Further confirmed analysis with online software IPA^® 8.7 and ELISA assay showed APOA1 and mTOR as potential biomarkers.

Conclusions: A distinct plasma proteomic profile may be associated with HSIL of Uyghur women.     

Metabolite profiles of interacting mycelial fronts differ for pairings of the wood decay basidiomycete fungus, Stereum hirsutum with its competitors Coprinus micaceus and Coprinus disseminatus

The paper presents the first proof-of principle study of metabolite profiles of the interacting mycelial fronts of a wood decomposer basidiomycete, Stereum hirsutum, paired with two competitor basidiomycetes, Coprinus disseminatus and C. micaceus, using TLC and GC-TOF-MS profiling. GC-TOF-MS profiles were information rich, with a total of 190 metabolite peaks detected and more than 120 metabolite peaks detected per sample. The metabolite profiles were able to discriminate between the interactions of S. hirsutum with the two species of Coprinus. In confrontation with C. micaceus, where S. hirsutum mycelial fronts always overgrew those of C. micaceus, there were down-regulations of metabolites in the interaction zone, compared to monocultures of both S. hirsutum and C. micaceus. In contrast, in pairings with C. disseminatus, whose mycelia overgrew those of S. hirsutum, there were some up-regulations compared with monoculture controls, the majority of the metabolites being characteristic of the S. hirsutum monoculture profile. These differences indicate that up-regulation of metabolites in the mycelia of S. hirsutum may be connected to a defensive role or to stress. The results also show proof of principle for the employment of metabolic profiling for biological discovery studies of metabolites produced by fungi that could be applied to natural product screening programmes.     

Biochemical,machine learning and molecular approaches for the differential diagnosis of Mucopolysaccharidoses

This study was aimed to construct classification and regression tree (CART) model of glycosaminoglycans (GAGs) for the differential diagnosis of Mucopolysaccharidoses (MPS). Two-dimensional electrophoresis and liquid chromatography–tandem mass spectrometry (LC–MS/MS) were used for the qualitative and quantitative analysis of GAGs. Specific enzyme assays and targeted gene sequencing were performed to confirm the diagnosis. Machine learning tools were used to develop CART model based on GAG profile. Qualitative and quantitative CART models showed 96.3% and 98.3% accuracy, respectively, in the differential diagnosis of MPS. The thresholds of different GAGs diagnostic of specific MPS types were established. In 60 MPS positive cases, 46 different mutations were identified in six specific genes. Among 31 different mutations identified in IDUA, nine were nonsense mutations and two were gross deletions while the remaining were missense mutations. In IDS gene, four missense, two frameshift, and one deletion were identified. In NAGLU gene, c.1693C?>?T and c.1914_1914insT were the most common mutations. Two ARSB, one case each of SGSH and GALNS mutations were observed. LC–MS/MS-based GAG pattern showed higher accuracy in the differential diagnosis of MPS. The mutation spectrum of MPS, specifically in IDUA and IDS genes, is highly heterogeneous among the cases studied.

     

The tissue- and developmental stage-specific involvement of autophagy genes in aggrephagy

ABSTRACT

Genetic screens have identified two sets of genes that act at distinct steps of basal autophagy in higher eukaryotes: the pan-eukaryotic ATG genes and the metazoan-specific EPG genes. Very little is known about whether these core macroautophagy/autophagy genes are differentially employed during multicellular organism development. Here we analyzed the function of core autophagy genes in autophagic removal of SQST-1/SQSTM1 during C. elegans development. We found that loss of function of genes acting at distinct steps in the autophagy pathway causes different patterns of SQST-1 accumulation in different tissues and developmental stages. We also identified that the calpain protease clp-2 acts in a cell context-specific manner in SQST-1 degradation. clp-2 is required for degradation of SQST-1 in the hypodermis and neurons, but is dispensable in the body wall muscle and intestine. Our results indicate that autophagy genes are differentially employed in a tissue- and stage-specific manner during the development of multicellular organisms.

Abbreviations: ATG: autophagy related; CLP: calpain family; EPG: ectopic PGL granules; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; LGG-1/LC3: LC3, GABARAP and GATE-16 family; MIT: microtubule interacting and transport; PGL: P granule abnormality protein; SQST-1: sequestosome-related; UPS: ubiquitin-proteasome system     

Ratios of leptin to insulin and adiponectin to endothelin are sex-dependently associated with extent of coronary atherosclerosis

Objective: Noninvasive diagnostics of early stages of coronary artery disease and discrimination between various extents of vascular lesions in patients is an important clinical problem especially considering wide spread use of cholesterol lowering drugs that affect lipid and lipoprotein profiling. The main goal of our study was to evaluate applicability of new combinations of noninvasive biomarkers such as leptin to insulin and adiponectin to endothelin ratios, for detection of early stages of coronary atherosclerosis versus later stages of the disease.

Patients and methods: A number of previously validated serum biomarkers were tested in a group of 500 patients with coronary artery disease and examined for their association with severity of coronary lesion according to Gensini score determined by coronary angiography.

Results: Lowest extent of coronary lesions was associated with significant increase in apoA-I levels and with significantly increased ratios of adiponectin to endothelin and leptin to insulin. In male but not in female patients, adiponectin to endothelin ratio below 7.0 was associated with Gensini score representing early to high coronary lesions (p?=?0.02). In female but not in male patients, leptin to insulin ratio below 3.5 was associated with Gensini score representing early to high coronary lesions (p?=?0.013).

Conclusion: Leptin to insulin and adiponectin to endothelin ratios are novel derived biomarkers useful for noninvasive diagnostics of initial stages of coronary lesions in patients with coronary artery disease.     

Methylated arginines and nitric oxide in end-stage renal disease: impact of inflammation,oxidative stress and haemodialysis

Abstract

Objectives: To determine whether inflammation (C-reactive protein, CRP), oxidative stress (malondialdehyde, MDA) or haemodialysis (HD) affect associations between asymmetric (ADMA), symmetric (SDMA) dimethylarginine, NG-monomethyl-L-arginine (L-NMMA) and nitrite/nitrate (NOx) in end-stage renal disease (ESRD).

Method: Metabolites were measured pre-HD, after 1 hour and end-HD in 40 ESRD patients (age 63?±?14 years).

Results: Positive associations between NOx and ADMA (p?=?0.04), SDMA (p?<?0.001) and L-NMMA (p?=?0.04) were observed pre-HD. Associations weakened during HD but were not significantly influenced by CRP or MDA.

Conclusions: HD, oxidative stress or inflammation did not significantly affect the positive associations between methylated arginines and NOx in ESRD.     

Label‐free profiling of skeletal muscle using high‐definition mass spectrometry

We report automated and time‐efficient (2 h per sample) profiling of muscle using ultra‐performance LC coupled directly with high‐definition MS (HDMS^E). Soluble proteins extracted from rat gastrocnemius (n = 10) were digested with trypsin and analyzed in duplicate using a 90 min RPLC gradient. Protein identification and label‐free quantitation were performed from HDMS^E spectra analyzed using Progenesis QI for Proteomics software. In total 1514 proteins were identified. Of these, 811 had at least three unique peptides and were subsequently used to assess the dynamic range and precision of LC‐HDMS^E label‐free profiling. Proteins analyzed by LC‐HDMS^E encompass the entire complement of glycolytic, β‐oxidation, and tricarboxylic acid enzymes. In addition, numerous components of the electron transport chain and protein kinases involved in skeletal muscle regulation were detected. The dynamic range of protein abundances spanned four orders of magnitude. The correlation between technical replicates of the ten biological samples was R² = 0.9961 ± 0.0036 (95% CI = 0.9940 – 0.9992) and the technical CV averaged 7.3 ± 6.7% (95% CI = 6.87 – 7.79%). This represents the most sophisticated label‐free profiling of skeletal muscle to date.     

Peripheral biomarkers of oxidative stress and their limited potential in evaluation of clinical features of Huntington's patients

Abstract

Context: Peripheral oxidative biomarkers could be useful for monitoring clinical features of Huntington's disease (HD).

Materials and methods: Cu/Zn-superoxide dismutase (Cu/Zn-SOD), neuron-specific enolase (NSE) and 8-hydroxy-2′-deoxyguanosine (8-oxoGua) serum levels were analysed in 18 HD patients and 10 controls. Clinical measures were recorded from each HD patients.

Results: Cu/Zn-SOD, NSE and 8-oxoGua values were higher in HD patients than in controls. Cu/Zn-SOD and NSE correlated positively. No correlation was observed between the biomarkers analysed and the clinical measures assessed.

Discussion and conclusion: Serum oxidative biomarkers could express the neuronal oxidative processes going on in HD patients but are inadequate to evaluate clinical features of the disease.