Cerebrospinal fluid proteome analysis reveals differentially abundant proteins in multiple sclerosis

Data processing and analysis have become true rate and success limiting factors for molecular research where a large number of samples of high complexity are included in the data set. In general rather complicated methodologies are needed for the combination and comparison of information as obtained from selected analytical platforms. Although commercial as well as freely accessible software for high-throughput data processing are available for most platforms, tailored in-house solutions for data management and analysis can provide the versatility and transparency eligible for e.g. method development and pilot studies.This paper describes a procedure for exploring metabolic fingerprints in urine samples from prostate and bladder cancer patients with a set of in-house developed Matlab tools. In spite of the immense amount of data produced by the LC-MS platform, in this study more than 10(10) data points, it is shown that the data processing tasks can be handled with reasonable computer resources. The preprocessing steps include baseline subtraction and noise reduction, followed by an initial time alignment. In the data analysis the fingerprints are treated as 2-D images, i.e. pixel by pixel, in contrast to the more common list-based approach after peak or feature detection. Although the latter approach greatly reduces the data complexity, it also involves a critical step that may obscure essential information due to undetected or misaligned peaks. The effects of remaining time shifts after the initial alignment are reduced by a binning and 'blurring' procedure prior to the comparative multivariate and univariate data analyses. Other factors than cancer assignment were taken into account by ANOVA applied to the PCA scores as well as to the individual variables (pixels). It was found that the analytical day-to-day variations in our study had a large confounding effect on the cancer related differences, which emphasizes the role of proper normalization and/or experimental design. While PCA could not establish significant cancer related patterns, the pixel-wise univariate analysis could provide a list of about a hundred 'hotspots' indicating possible biomarkers. This was also the limited goal for this study, with focus on the exploration of a really huge and complex data set. True biomarker identification, however, needs thorough validation and verification in separate patient sets. (c) 2011 Elsevier B.V. All rights reserved.

In the northern hemisphere moose has been found to be suitable as a monitoring animal for the presence of cadmium in the environment. The metal accumulates mainly in the kidney and the liver, with the rate of accumulation dependent on age and possibly also on gender. Collection of tissue material often results in sample selections with disparate age and gender composition, which makes comparison between different regions and different studies difficult. A previous large scale investigation of metals in kidney and liver from moose in Sweden provided Cd data (n = 3,817 and 3,802, respectively) to further explore the relation between Cd accumulation and age/gender. Based on local averages, the individual deviations were analysed with respect to the factors age and gender resulting in an ‘ageing function’ for each gender and organ. In addition, estimates of the pure individual variations were obtained; the standard deviations correspond to a factor 1.7–1.9 for the Cd concentration, which indicates that 25–30 samples are needed to give a representative mean value (with RSD ≈10%). In order to be able to compare results from different studies, all individual results can be transformed to represent a ‘standard moose’ with respect to age and gender. A comparison along these lines was undertaken between Cd levels in Alaska and Sweden. Finally, a relationship between the Cd levels in kidney and liver was derived, providing at least rough estimates for kidney from liver values (or vice versa).

Metabolomic fingerprinting is a growing strategy for characterizing complex biological samples without detailed prior knowledge about the metabolic system. A two-way analysis system with liquid separation and mass spectrometric detection provides detail-rich data suitable for such fingerprints. As a model study, human urine samples, obtained after intake of coffee, tea, or water, were analyzed with capillary electrophoresis electrospray ionization time-of-flight mass spectrometry (CE-ESI-TOF-MS). In-house-developed software (in Matlab) was utilized to manage and explore the large amount of data acquired (230 CE-MS runs, each with 50-100 million nonzero data points). After baseline and noise reduction, followed by suitable binning in time and m/z, the data sets comprised 9 and 14 million data points in negative and positive ESI mode, respectively. Finally, a signal threshold was applied, further reducing the number to about 100 000 data points per data set. A set of interactive exploratory tools, utilizing principal component analysis (PCA) and analysis of variance (ANOVA) results based on a general linear model, facilitated visual interpretation with score plots (for group assessment) and differential fingerprints (for "hot spot" detection). In the model study highly significant differences due to beverage intake were obtained among the 10 first principal components (p < 10(-6) for two of the components in both ESI modes). Especially, the contrasts between "coffee" and "tea or water" indicated several "hot spots" with highly elevated intensities (e.g., for uncharged masses 93, 94, 109, 119, 123, 132, 148, 169, 178, 187, 190, and 193) suitable for further analysis, for example, with tandem MS.

Hyphenated techniques such as capillary electrophoresis-mass spectrometry (CE-MS) or high-performance liquid chromatography with diode array detection (HPLC-DAD), etc., are known to produce a huge amount of data since each sample is characterized by a two-way data table. In this paper different ways of obtaining sample-related information from a set of such tables are discussed. Working with original data requires alignment techniques due to time shifts caused by unavoidable variations in separation conditions. Other pre-processing techniques have been suggested to facilitate comparison among samples without prior peak alignment, for example, 'binning' and/or 'blurring' the data along the time dimension. All these techniques, however, require optimization of some parameters, and in this paper an alternative parameter-free method is proposed. The individual data tables (X) are represented as Gram matrices (XXT), where the summation is taken over the time dimension. Hence the possible variations in time scale are eliminated, while the time information is at least partly preserved by the correlation structure between the detection channels. For comparison among samples, a similarity matrix is constructed and explored by principal component analysis and hierarchical clustering. The Gram matrix approach was tested and compared to some other methods using 'binned' and 'blurred' data for a data set with CE-MS runs on urine samples. In addition to data exploration by principal component analysis and hierarchical clustering, a discriminant partial least squares model was constructed to discriminate between the samples that were taken with and without the prior intake of a drug. The result showed that the proposed method is at least as good as the others with respect to cluster identification and class prediction. A distinct advantage is that there is no need for parameter optimization, while a potential drawback is the large size of the Gram matrices for data with high mass resolution.

Today a high degree of "false" appendicitis diagnoses are occurring. In this study, a screening experiment of biomarkers of two different kinds of appendicitis, gangrenous and phlegmonous, were conducted with CE and CEC coupled to MS. Plasma samples were obtained from patients pre- and post-surgery. A large amount of data was generated to be able to compare them, and chemometrics tools were utilized to visualize the differences. Indicative patterns were found for both pre- and post-surgery of the two types of inflammation as well as between them. The divergences were traced back to the MS peaks obtained in the CE- and CEC-MS setups as possible biomarkers for the two forms of appendicitis.

Purpose: To investigate lymphatic drainage as measured by lymphoscintigraphy in the arms of patients undergoing either sentinel lymph node biopsy (SNB) or axillary lymph node dissection (ALND).Material and Methods: From January 2001 to December 2002, 30 patients with unilateral invasive breast carcinoma underwent breast-conserving surgery with SNB and 30 patients with ALND. All patients received radiotherapy to the breast. Lymphoscintigraphy was performed, and skin circulation, skin temperature, and arm volume were measured 2 - 3 years after radiotherapy.Results: None of the 30 patients who underwent SNB showed any clinical manifestation of lymphedema. Of the 30 patients undergoing ALND, six (20%) had clinical lymphedema, with an arm volume that was > 10% larger on the operated than on the non-operated side ( P < 0.01). Scintigraphically, visual analysis revealed lymphatic dysfunction in three patients, manifested as forearm dermal back flow. Two of these patients also had an increased arm volume. Quantitative analysis showed no differences between the groups, apart from a smaller amount of isotope in the axilla in the ALND group. There was no difference in skin circulation or skin temperature.Conclusion: Our study shows that lymph drainage in the operated arm compared with the non-operated arm was less affected by SNB than by ALND, and that morbidity associated with SNB was lower than with ALND. However, the results do not confirm our hypothesis that lymphoscintigraphy can reveal differences in lymph circulation that are not evident clinically in the form of manifest lymphedema. The most sensitive clinical method of assessing lymph drainage seems to be measurement of arm volume.

Background and Aims: In patients with primary hyperparathyroidism (PHPT), parathyroid imaging is nowadays routinely used for the purpose to perform a focused unilateral minimally invasive operation. The outcome of this new strategy has, however, not been established in randomised trials. Material and Methods: Patients were randomised to either preoperative localisation with sestamibi scintigraphy and ultrasonography (group I) or no preoperative localisation (group II). In group I, a minimally invasive parathyroidectomy was performed in patients in whom both localisation studies were consistent with a single pathological gland, whereas a conventional bilateral neck exploration was performed in cases with negative localisation findings. In group II all patients underwent conventional bilateral neck exploration. Primary outcome measure was normocalcaemia at 6 months postoperatively. Results: In the preoperative localisation group (group I) 23/50 (46%) of the patients could be operated on with the focused operation whereas 26/50 (52%) were operated on by bilateral neck exploration. All patients in the no localisation group (group II; n=50) were operated on with the intended bilateral neck operation. Normocalcaemia was obtained in 96% and 94% in group I and II, respectively. Total (localisation and operative) costs were 21% higher in group I. Conclusions: Routine preoperative localisation, with the intention to perform minimally invasive parathyroidectomy, is not cost effective if concordant results of scintigraphy and ultrasonography are a prerequisite for the focused operation. Less than half of the patients were successfully managed with this strategy, at a higher cost and without obtaining a more favourable clinical outcome.

Comparing CE-MS fingerprints of urine samples, obtained after intake of coffee, tea or water

The etch rate and surface roughness of polished and lapped AT-cut quartz subjected to hot (90, 110, and 130 degrees C), concentrated (50, 65, 80 wt %) ammonium bifluoride have been investigated. Having used principal component analysis to verify experimental solidity and analyze data, we claim with confidence that this parameter space does not, as elsewhere stated, allow for a polishing effect or even a preserving setting. Etch rates were found to correlate well, and possibly logarithmically, with temperature except for the hottest etching applied to lapped material. Roughness as a function of temperature and concentration behaved well for the lapped material, but lacked systematic variation in the case of the polished material. At the lowest temperature, concentration had no effect on etch rate or roughness. Future efforts are targeted at temperatures and concentrations closer to the solubility limit.

Peptide mass fingerprints were obtained for three different proteins using three different digestion procedures in triplicates with liquid chromatography coupled to electrospray ionization mass spectrometry. For each protein the results were compared with multivariate data analysis (cluster analysis, kernel principal component analysis) and pair-wise contrast evaluation. Clear systematic differences between the digestion procedures were established for all the proteins. The visual presentation of the pair-wise differences between procedures could to some extent be related to the protein fragments, although the main objective was to identify m/z and retention regions in the original peptide maps that should be subject to further exploration.

A method to prepare NaOH sediment extracts for organic P compound analysis with electrospray ionization tandem mass spectrometry (ESI-MS-MS) was developed on natural samples. Ion exchange, rotary evaporation and mass cut-off filtering proved to be suitable for sample preparation. Samples were analyzed with ESI-MS-MS, and reproducibility and repeatability of the method was calculated. In addition, (31)P-nuclear magnetic resonance spectroscopy ((31)P NMR) was used to measure recovery of different P compound groups such as orthophosphate (Ortho-P), orthophosphate monoesters (Monoester-P), orthophosphate diesters (Diester-P) and pyrophosphates (Pyro-P). The developed sample preparation method resulted in an easy-to-spray liquid for the ESI-MS-MS instrumentation. The overall P recovery was 65% and (31)P NMR showed that Diester-P, possibly in the form of DNA, was apparently lost through the filtering step most likely due to their size. Variances in the total intensities of the MS scans (relative standard deviation (R.S.D.) 35-54%) were for about 50% due to repeated MS runs. Covariances of the peaks in the MS spectra were calculated to be for about 30% due to the sample preparation procedure. Finally, with the ESI-MS-MS approach, 11 peaks in the mass spectra were found likely to represent phosphate containing compounds.

Aim: As a means of staging the axilla with minimal surgical trauma, sentinel lymph node biopsy (SNB) has dramatically altered the management of early-stage breast cancer. The aim of this prospective multicentre study was to assess the safety of the method in cases of non-palpable tumours and in cases with an open biopsy prior to SNB.Method: In the period 1999-2001, 57 non-palpable breast cancers and 75 patients with diagnostic biopsy were collected prospectively to the first part of the study. In the second part, 745 patients with non-palpable breast cancers and 86 cases with prior open surgery diagnosed between 2000 and 2005 were followed up till the end of 2005. All patients in the first part of the study had an axillary clearance irrespective of sentinel node status, whereas in the second part axillary clearance was done only if the sentinel node was metastatic.Results: The detection rate was 95% in the group of non-palpable breast cancers, with a false negative rate of 5.6% (1/18), and the corresponding figures for the group with prior intervention were 96% and 10% (2/20). Two axillary recurrences, after a negative SNB at primary surgery, were found in the non-palpable group after 16 and 17 months, respectively. No axillary recurrence has been observed in the group of cancers with a prior open biopsy. Four women in the non-palpable group and two women with a diagnostic operation experienced distant metastases.Conclusion: We conclude that SNB is a safe procedure for women with non-palpable breast cancer, as well as after previous open diagnostic excision. (c) 2006 Elsevier Ltd. All rights reserved.

Several studies have shown that deficient uptake or excessive break down of membrane phospholipids may be associated with neurodegenerative and psychiatric disorders. The purpose of the present study was to examine the effects of postnatal iron administration in lipid composition and behavior and whether or not the established effects may be altered by subchronic administration of the neuroleptic compounds, clozapine and haloperidol. In addition to motor activities such as locomotion, rearing and activity, a targeted lipidomics approach has been used to investigated the brains of eight groups of mice (four vehicle groups and four iron groups) containing six individuals in each group treated with vehicle, low dose clozapine, high dose clozapine and haloperidol. Lipids were extracted by the Folch method and analyzed using reversed‐phase capillary liquid chromatography coupled on‐line to electrospray ionization mass spectrometry (LC/ESI/MS). Identification of phosphatidylcholine (PC) and sphingomyelin (SM) molecular species was based on their retention time, m / z ratio, head group specific up‐front fragmentation and analysis of the product ions produced upon fragmentation. A comparison between the Ve‐groups and Fe‐groups showed that levels of PC and SM molecular species and motor activities were significantly lower in Fe‐Ve compared to Ve‐Ve. The effects of neuroleptic treatment with and without iron supplementation were studied. In conclusion our results support the hypothesis that an association between psychiatric disorders and lipid and behavior abnormalities in the brain exists.

This work presents the development of a general and fast method for metabolic profiling of urine, using capillary electrophoresis-electrospray ionisation mass spectrometry (CE-ESIMS) and multivariate data analysis (DA). Human urine samples collected before and after ingestion of paracetamol were analysed at acidic and basic CE conditions, using both positive and negative ESI-MS detection. Analysis of the entire resulting data set, with no prior knowledge of the target compounds, using pair-wise 'fuzzy' correlation and eigenvalue analysis enabled the samples to be discriminated between on the basis of blank urine and urine collected after drug intake. By generating two-dimensional loadings plots, it was also possible to identify the m/z values of the substances responsible for the differentiation between control and dosed samples.

A stepwise study of common factors for the extraction of acrylamide (AA) from relevant food matrices was performed. The investigated extraction factors were sample particle size (fine or coarse), defatting (yes or no), extraction solvent (water or water/methanol), homogenisation by Ultra Turrax (yes or no), extraction temperature (25 or 60°C) and extraction time (5min to 17h). An optimised method comprised the use of fine particles (<1000μm), water as the extraction solvent and shaking of the sample for 45min at 25°C. This extraction method was suitable for all tested matrices (coffee, crispbread, mashed potatoes, milk chocolate and potato crisps). The analytical results (from LC-MS/MS analysis after SPE clean-up) correlated well with those obtained by the original, more labour-intensive, extraction procedure and there was excellent agreement with the assigned AA levels of several proficiency test samples analysed for evaluation. Defatting and additional homogenisation by Ultra Turrax did not increase the AA yield when other factors were set to appropriate levels. In general, the study indicates that incomplete extraction is the most likely cause of erroneous results. This might occur when the food is not sufficiently macerated, when water/methanol is used as the extraction solvent, and when using a short extraction time or low extraction temperature, especially when these conditions are combined. Formation of AA during the extraction procedure is another possible error source, though it was not seen under any of the experimental conditions employed when a fructose-enriched blank potato tissue was extracted.