A method for improving SELDI-TOF mass spectrometry data quality

Background  

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a powerful tool for rapidly generating high-throughput protein profiles from a large number of samples. However, the events that occur between the first and last sample run are likely to introduce technical variation in the results.     

Comparison of algorithms for pre-processing of SELDI-TOF mass spectrometry data

MOTIVATION: Surface-enhanced laser desorption and ionization (SELDI) time of flight (TOF) is a mass spectrometry technology. The key features in a mass spectrum are its peaks. In order to locate the peaks and quantify their intensities, several pre-processing steps are required. Though different approaches to perform pre-processing have been proposed, there is no systematic study that compares their performance. RESULTS: In this article, we present the results of a systematic comparison of various popular packages for pre-processing of SELDI-TOF data. We evaluate their performance in terms of two of their primary functions: peak detection and peak quantification. Regarding peak quantification, the performance of the algorithms is measured in terms of reproducibility. For peak detection, the comparison is based on sensitivity and false discovery rate. Our results show that for spectra generated with low laser intensity, the software developed by Ciphergen Biosystems (ProteinChip Software 3.1 with the additional tool Biomarker Wizard) produces relatively good results for both peak quantification and detection. On the other hand, for the data produced with either medium or high laser intensity, none of the methods show uniformly better performances under both criteria. Our analysis suggests that an advantageous combination is the use of the packages MassSpecWavelet and PROcess, the former for peak detection and the latter for peak quantification.     

The application of SELDI-TOF mass spectrometry to mammalian cell culture

Surface Enhanced Laser Desorption/Ionisation Time-of-Fight Mass Spectrometry (SELDI-TOF MS) is a technique by which protein profiles can be rapidly produced from a wide variety of biological samples. By employing chromatographic surfaces combined with the specificity and reproducibility of mass spectrometry it has allowed for profiles from complex biological samples to be analysed. Profiling and biomarker identification have been employed widely throughout the biological sciences. To date, however, the benefits of SELDI-TOF MS have not been realised in the area of mammalian cell culture. The advantages in identifying markers for cell stresses, apoptosis and other culture parameters mean that these tools could help greatly to enhance monitoring and control of bioreaction process and improve the production of therapeutics. Better characterisation of culture systems through proteome analysis will allow for improved productivity and better yields.     

Regression analysis and modelling of data acquisition for SELDI-TOF mass spectrometry

MOTIVATION: Pre-processing of SELDI-TOF mass spectrometry data is currently performed on a largel y ad hoc basis. This makes comparison of results from independent analyses troublesome and does not provide a framework for distinguishing different sources of variation in data. RESULTS: In this article, we consider the task of pooling a large number of single-shot spectra, a task commonly performed automatically by the instrument software. By viewing the underlying statistical problem as one of heteroscedastic linear regression, we provide a framework for introducing robust methods and for dealing with missing data resulting from a limited span of recordable intensity values provided by the instrument. Our framework provides an interpretation of currently used methods as a maximum-likelihood estimator and allows theoretical derivation of its variance. We observe that this variance depends crucially on the total number of ionic species, which can vary considerably between different pooled spectra. This variation in variance can potentially invalidate the results from naive methods of discrimination/classification and we outline appropriate data transformations. Introducing methods from robust statistics did not improve the standard errors of the pooled samples. Imputing missing values however-using the EM algorithm-had a notable effect on the result; for our data, the pooled height of peaks which were frequently truncated increased by up to 30%.     

Quadratic variance models for adaptively preprocessing SELDI-TOF mass spectrometry data

Background  

Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI) is a proteomics tool for biomarker discovery and other high throughput applications. Previous studies have identified various areas for improvement in preprocessing algorithms used for protein peak detection. Bottom-up approaches to preprocessing that emphasize modeling SELDI data acquisition are promising avenues of research to find the needed improvements in reproducibility.     

Profiling of apoptotic changes in human breast cancer cells using SELDI-TOF mass spectrometry.

Apoptosis is a key process in the response of tumours to chemotherapeutic agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many tumor cells, while sparing most normal cells. Several chemotherapeutic drugs synergize with TRAIL in reducing tumor growth and inducing apoptosis. Because some tumour cells respond poorly to these treatments, biomarkers that predict clinical responsiveness are needed. This study used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify novel apoptotic markers in TRAIL and etoposide (T+E)-treated MDA-MB-231 and ZR-75-1 breast cancer cells and MCF-10A non-transformed breast cells. T+E induced apoptosis, increasing caspase-3 activity at 4-8h, in all cell lines. Protein profiles revealed two prominent peaks, m/z 10090 and 8560, which decreased significantly during apoptosis. Mass spectrometry sequencing of tryptic peptides identified these proteins as S100A6 (confirmed immunologically) and ubiquitin (confirmed against a purified standard), respectively. Caspase inhibition prevented the decrease in both proteins during T+E-induced apoptosis whereas proteasome inhibition combined with T+E further decreased ubiquitin, possibly by preventing its recycling. Using SELDI-TOF MS we have identified S100A6 and ubiquitin as potential protein markers of apoptosis. Further validation using patient samples is required to confirm their potential utility in monitoring the effectiveness of anti-cancer drugs in inducing tumour cell apoptosis.     

Human Endothelial Progenitor Cells Internalize High-Density Lipoprotein

Endothelial progenitor cells (EPCs) originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL), and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate), cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of “strings of pearl”- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568–treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal intraellular pathway and accumulated prominently in all parts of the Golgi apparatus and in lipid droplets. Subsequently, also the RER and mitochondria were involved. These studies demonstrated the different intracellular pathway of HDL-derived bodipy-cholesterol and HDL-derived bodipy-cholesteryl oleate by EPCs, with concomitant.     

Quantitative quality-assessment techniques to compare fractionation and depletion methods in SELDI-TOF mass spectrometry experiments

Motivation: Mass spectrometry (MS), such as the surface-enhancedlaser desorption and ionization time-of-flight (SELDI-TOF) MS,provides a potentially promising proteomic technology for biomarkerdiscovery. An important matter for such a technology to be usedroutinely is its reproducibility. It is of significant interestto develop quantitative measures to evaluate the quality andreliability of different experimental methods. Results: We compare the quality of SELDI-TOF MS data using unfractionated,fractionated plasma samples and abundant protein depletion methodsin terms of the numbers of detected peaks and reliability. Severalstatistical quality-control and quality-assessment techniquesare proposed, including the Graeco–Latin square designfor the sample allocation on a Protein chip, the use of thepairwise Pearson correlation coefficient as the similarity measurebetween the spectra in conjunction with multi-dimensional scaling(MDS) for graphically evaluating similarity of replicates andassessing outlier samples; and the use of the reliability ratiofor evaluating reproducibility. Our results show that the numberof peaks detected is similar among the three sample preparationtechnologies, and the use of the Sigma multi-removal kit doesnot improve peak detection. Fractionation of plasma samplesintroduces more experimental variability. The peaks detectedusing the unfractionated plasma samples have the highest reproducibilityas determined by the reliability ratio. Availability: Our algorithm for assessment of SELDI-TOF experimentquality is available at http://www.biostat.harvard.edu/~xlin Contact: harezlak{at}post.harvard.edu Supplementary information: Supplementary data are availableat Bioinformatics online. Associate Editor: Thomas Lengauer     

High-Density Lipoprotein Function in Exudative Age-Related Macular Degeneration

PurposeHigh-density lipoproteins (HDL) have long been implicated in the pathogenesis of age-related macular degeneration (AMD). However, conflicting results have been reported with regard to the associations of AMD with HDL-cholesterol levels. The present study is the first to assess HDL composition and metrics of HDL function in patients with exudative AMD and control patients.MethodsBlood samples were collected from 29 patients with exudative AMD and 26 age-matched control patients. Major HDL associated apolipoproteins were determined in apoB-depleted serum by immunoturbidimetry or ELISA, HDL-associated lipids were quantified enzymatically. To get an integrated measure of HDL quantity and quality, we assessed several metrics of HDL function, including cholesterol efflux capacity, anti-oxidative and anti-inflammatory activities using apoB-depleted serum from study participants.ResultsIn our study, we observed that the HDL associated acute phase protein serum amyloid A (SAA) was significantly increased in AMD patients (p<0.01), whereas all other assessed apolipoproteins including ApoA-I, apoA-II, apoC-II, apoC-III and apoE as well as major HDL associated lipids were not altered. HDL efflux capacity, anti-oxidative capacity and arylesterase activity were not different in AMD patients when compared with the control group. The ability of apoB-depleted serum to inhibit monocyte NF-κB expression was significantly improved in AMD patients (mean difference (MD) -5.6, p<0.01). Moreover, lipoprotein-associated phospholipase A2 activity, a marker of vascular inflammation, was decreased in AMD subjects (MD -24.1, p<0.01).ConclusionsThe investigated metrics of HDL composition and HDL function were not associated with exudative AMD in this study, despite an increased content of HDL associated SAA in AMD patients. Unexpectedly, anti-inflammatory activity of apoB-depleted serum was even increased in our study. Our data suggest that the investigated parameters of serum HDL function showed no significant association with exudative AMD. However, we cannot exclude that alterations in locally produced HDL may be part of the AMD pathogenesis.     

ABCA1在动脉粥样硬化发生与发展中的作用

腺苷三磷酸结合盒转运体A1(ATP binding cassette transporter A1 ,ABCA1)是一种整合膜蛋白,它以ATP为能源,促进细胞内游离胆固醇和磷脂的流出,在胆固醇逆转运(RCT)和HDL生成的起始步骤中起重要作用,被称作RCT守门人。核受体PPARs、LXRs和FXR对ABCA1蛋白的表达具有调控作用。人体50种组织中存在有ABCA1 mRNA,在胰、肝、肺、肾上腺和胎儿组织中ABCAl表达水平最高,ABCAl功能障碍将导致巨噬细胞内大量的胆固醇沉积而成为泡沫细胞,继而漫润血管壁,促进As的发生发展。     

High-Density Lipoprotein Aggregated by Oxidation Induces Degeneration of Neuronal Cells

Abstract: We have previously reported that high-density lipoprotein (HDL) exhibits antineuritogenic effects on chicken cerebral cells in culture. In the present study, we show the effects of HDLs, oxidized by UV irradiation or heating, on chicken cerebral neurons in culture. Both treatments produced several physical and chemical changes in the HDLs, i.e., formation of lipid peroxides, enlargement of HDL diameters, an increased exposure of the tryptophan groups of the apolipoprotein A-I to a more hydrophilic environment, formation of bityrosines, and cross-linking of apolipoprotein A-I. When these treatments were performed in the absence of EDTA, most of the modifications described above were more intense and HDLs formed a macroaggregate that displays a rosette-like structure. The aggregated HDLs produced neurodegeneration and death when added to both undifferentiated and differentiated cerebral neurons in culture. This process was accompanied by the disorganization of the cellular microtubular cytoskeleton and hyperphosphorylation of the microtubule-associated protein tau. Native HDL or HDLs treated in the presence of EDTA inhibited the neuritogenesis of undifferentiated neurons but did not show any significant effect on the differentiated neurons in culture. The effects on the cellular cytoskeleton and morphology of aggregated HDLs recall those of the fibrillar β-amyloid peptide. The present results suggest that aggregated HDLs could participate in neurodegeneration associated with oxidative stress in the CNS.     

Paradoxical Severe Decrease in High-Density Lipoprotein Cholesterol Due to Rosiglitazone-Fenofibrate Interaction

ObjectiveTo determine whether the marked decrease in high-density lipoprotein cholesterol (HDL-C) occasion- ally associated with combination fibrate-thiazolidinedione therapy results from interaction between the 2 drugs or is solely the result of fibrate administration, a previously rec- ognized cause.MethodsWe prospectively followed the clinical course of 2 patients receiving fenofibrate and rosiglitazone and reviewed the relevant literature, searching PubMed for reports describing striking reductions in HDL-C associ- ated with fibrate administration alone and in conjunction with rosiglitazone and statins. Additional references were obtained from the bibliography of each identified article.ResultsEach of the 2 patients demonstrated a Drug Interaction Probability Score score of 9, indicating a highly probable likelihood of interaction. Critical review of all reported cases of concurrent fenofibrate-rosiglitazone– associated decreases in HDL-C failed to show conclusive evidence that the HDL-C decrease could be due to an inter- action between the 2 drugs as opposed to either drug being given alone.ConclusionsIn at least some patients who experience marked HDL-C decrease when given a combination of fenofibrate and rosiglitazone, this severe adverse effect is the result of a drug interaction between the 2 pharmaceuti- cal agents and is not reproduced by the administration of either drug singly. (Endocr Pract. 2010;16:382-388)     

SELDI-TOF mass spectra: a view on sources of variation

Adequate interpretation of mass spectrometry data can yield valuable biomarkers. However, spectrum interpretation is a complicated task. This paper reviews the various factors that determine a sample's spectrum and demonstrates the role of these factors in the interpretation process. We derive a simulation model that adequately predicts the expected spectrum based on known sample content and, in the reverse mode, obtain an analysis model that adequately fits an observed spectrum based on the hypothesized sources of variation.     

基于高密度脂蛋白代谢与重塑的抗动脉粥样硬化研究及相关药物

动脉粥样硬化（atherosclerosis,AS）是一种主要因血脂代谢紊乱引发的慢性炎症性血管疾病,以血管内膜下巨噬细胞和血管平滑肌细胞过度蓄脂泡沫化为主要病理特征。高密度脂蛋白（high-density lipoprotein,HDL）通过胆固醇逆向转运（reverse cholesterol transport,RCT）将外周细胞中的胆固醇运输到肝脏然后经胆汁排出体外,从而改善血脂水平和细胞的过度蓄脂,被认为是HDL抗AS的基础。然而,大量流行病学证据表明,虽然血浆高密度脂蛋白胆固醇（high-density lipoprotein cholesterol,HDL-C）水平与心血管风险呈负相关,但仅仅提高HDL-C水平的治疗策略不一定能增加临床效益。因此,学术界认识到HDL水平不足以反映其RCT能力,而更多取决于HDL功能。本文综述了参与调节HDL功能的各种分子对HDL代谢与重塑过程的影响,以及针对上述过程的相关药物研究进展,为更全面评价HDL的抗AS作用提供理论参考。     

High-Density Lipoprotein Prevents Endoplasmic Reticulum Stress-Induced Downregulation of Liver LOX-1 Expression

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a specific cell-surface receptor for oxidized-low-density lipoprotein (ox-LDL). The impact of high-density lipoprotein (HDL) on endoplasmic reticulum (ER) stress-mediated alteration of the LOX-1 level in hepatocytes remains unclear. We aimed to investigate the impact on LOX-1 expression by tunicamycin (TM)-induced ER stress and to determine the effect of HDL on TM-affected LOX-1 expression in hepatic L02 cells. Overexpression or silencing of related cellular genes was conducted in TM-treated cells. mRNA expression was evaluated using real-time polymerase chain reaction (PCR). Protein expression was analyzed by western blot and immunocytochemistry. Lipid uptake was examined by DiI-ox-LDL, followed by flow cytometric analysis. The results showed that TM induced the upregulation of ER chaperone GRP78, downregulation of LOX-1 expression, and lipid uptake. Knock down of IRE1 or XBP-1 effectively restored LOX-1 expression and improved lipid uptake in TM-treated cells. HDL treatment prevented the negative impact on LOX-1 expression and lipid uptake induced by TM. Additionally, 1–10 μg/mL HDL significantly reduced the GRP78, IRE1, and XBP-1 expression levels in TM-treated cells. Our findings reveal that HDL could prevent the TM-induced reduction of LOX-1 expression via inhibiting the IRE1/XBP-1 pathway, suggesting a new mechanism for beneficial roles of HDL in improving lipid metabolism.     

Increased Plasma Apolipoprotein E-Rich High-Density Lipoprotein and Its Effect on Serum High-Density Lipoprotein Cholesterol Determination in Patients with Familial Hyperalphalipoproteinemia Due to Cholesteryl Ester Transfer Activity Deficiency

Plasma apo E-rich HDL was studied in regard to its quantity and chemical composition in the members of a family with cholesteryl ester transfer activity deficiency, exhibiting familial hyperalphalipoproteinemia. The approach involved a simple precipitation method established in our laboratory. Serum apo E-rich HDL concentrations for two homozygous members were elevated up to 66 and 60 mg/dl in terms of cholesterol (normal, 6.7 ± 2.3 mg/dl, n = 38), and to 9.4 and 10.8 mg/dl in terms of apo E (normal, 2.6 ± 1.5 mg/dl, n = 38). The cholesterol/apo E ratio (mole/mole) of apo E-rich HDL was higher in two homozygotes (669 and 531) than in two cholestatic patients with elevated apo E-rich HDL (268 and 149) and in normal subjects (242 ± 115, n = 38). Chromatographic studies of the serum from a homozygote showed enlargement of all HDL subclasses and apo E in the larger HDL subclass. These facts mndicate that the increase of apo E-rich HDL in this disease occurs secondarily to the enlargement of HDL particles, which require substances to cover their cores, having expanded due to the accumulation of cholesteryl ester. The sera from the homozygotes gave HDL cholesterol concentrations which were remarkably discrepant among commercial precipitating reagents, because of the difference in recovery of apo E-rich HDL with these reagents.     

Trypanosome Lytic Factor,an Antimicrobial High-Density Lipoprotein,Ameliorates Leishmania Infection

Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF) is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system.