Alamgir Khan

High-abundance protein depletion : comparison of methods for human plasma biomarker discovery

Affinity depletion of abundant proteins from human plasma has become a routine sample preparation strategy in proteomics used prior to protein identification and quantitation. To date, there have been limited published studies comparing the performance of commercially available depletion products. We conducted a thorough evaluation of six depletion columns using 2‐DE combined with sophisticated image analysis software, examining the following criteria: (i) efficiency of high‐abundance protein depletion, (ii) non‐specific removal of other than the targeted proteins and (iii) total number of protein spots detected on the gels following depletion. From all the products investigated, the Seppro IgY system provided the best results. It displayed the greatest number of protein spots on the depleted plasma gels, minimal non‐specific binding and high efficiency of abundant protein removal. Nevertheless, the increase in the number of detected spots compared with the second best performing and cheaper multiple affinity removal column (MARC) was not shown to be statistically significant. The ProteoPrep spin column, considered to be the “deepest” depletion technique available at the time of conducting the study, surprisingly displayed significantly fewer spots on the flow‐through fraction gels compared with both the Seppro and the MARC. The spin column format and low plasma capacity were also found to be impractical for 2‐DE. To conclude, we succeeded in providing an overview of the depletion columns performances with regard to the three examined areas. Our study will serve as a reference to other scientists when deciding on the optimal product for their particular projects.

Infection of Meloidogyne javanica by Paecilomyces lilacinus

The in vitro interaction of Paecilomyces lilacinus strain 251 with eggs, 3rd and 4th stage juveniles and adult females of Meloidogyne javanica was studied. Eggs of all stages, including those containing unhatched juveniles, were infected by P. lilacinus. Infection of eggs occurred following flattening of hyphae to the egg surface and formation of appressoria. Sometimes these occurred within extensive networks of hyphae of the egg surface. Hyphae later grew out of the egg to continue growing or form conidiophores. Third and 4th stage juveniles and adult females were readily infected, with hyphae and conidiophores penetrating the body wall. Die Infektion von Meloidogyne javanica durch Paecilomyces lilacinus - Es wurden die in vitro auftretenden Wechselwirkungen zwischen Paecilomyces lilacinus Stamm 251 und den Eiern, J3, J4 und adulten Weibchen von Meloidogyne javanica untersucht. Eier wurden in allen Stadien von P. lilacinus infiziert einschliesslich der ungeschlupfte J2 enthaltenden Eier. Die Infektion erfolgte anschliessend an eine Abflachung von Hyphen auf der Eioberflache und eine Appressorienbildung. Manchmal erschienen diese innerhalb eines ausgedehnten Netzwerkes von Hyphen auf der Eioberflache. Spater wuchsen Hyphen aus dem Ei heraus, wuchsen weiter oder bildeten Konidiophoren. Juvenile des dritten oder vierten Stadiums und adulte Weibchen wurden ohne weiteres befallen, wobei Hyphen und Konidiophoren durch die Korperwand drangen.

Guidelines for reporting the use of gel electrophoresis in proteomics

Gibson, Frank Anderson, Leigh Babnigg, Gyorgy Baker, Mark Berth, Matthias Binz, Pierre-Alain Borthwick, Andy Cash, Phil Day, Billy W. Friedman, David B. Garland, Donita Gutstein, Howard B. Hoogland, Christine Jones, Neil A. Khan, Alamgir Klose, Joachim Lamond, Angus I. Lemkin, Peter F. Lilley, Kathryn S. Minden, Jonathan Morris, Nicholas J. Paton, Norman W. Pisano, Michael R. Prime, John E. Rabilloud, Thierry Stead, David A. Taylor, Chris F. Voshol, Hans Wipat, Anil Jones, Andrew R. 2 NATURE PUBLISHING GROUP NEW YORK 335WX

Detection and quantitation of forty eight cytokines, chemokines, growth factors and nine acute phase proteins in healthy human plasma, saliva and urine.

Cytokines, chemokines, growth factors (CCGFs) and other low abundance proteins/peptides in human body fluids or in tissues are potential biomarkers. Human body fluids such as plasma, saliva, urine, etc. are being analyzed more frequently than tissues primarily because of ease of sample collection. However, available information on concentrations of a large number of CCGFs in various body fluids of the same healthy individuals and gender-specific CCGFs is limited. In this work concentrations of 48 CCGFs were measured using multiplex bead assays and compared between plasma, saliva and urine collected from 20 male and female healthy volunteers. Forty three CCGFs were detected at least in one sample type of which 37 were in plasma, 41 were in saliva, and 34 were in urine; five CCGFs were not detected in any sample. Concentrations of detected CCGFs differed significantly between sample types but similar between gender groups. Gender-specific CCGFs were also observed. Concentrations of nine acute phase proteins were also measured from plasma, saliva and urine to determine general health conditions of the volunteers. This work will provide an idea of which CCGFs are detectable and their relative concentrations in healthy human plasma, saliva and urine and which CCGFs are gender-specific.

Discovery of a novel biomarker in the urine in women with endometriosis

OBJECTIVE To investigate whether proteins secreted in urine differ between women with and without endometriosis. DESIGN Laboratory study using human urine. SETTING University-based laboratory. PATIENT(S) Women with and without endometriosis undergoing laparoscopy, hysteroscopy and curettage. INTERVENTION(S) Urine collection from women with and without endometriosis. MAIN OUTCOME MEASURE(S) Proteomic techniques and mass spectrometry to identify proteins secreted in the urine of women with and without endometriosis. RESULT(S) On average, 133 proteins were significantly different between women with and without endometriosis. Cytokeratin-19 was highly up-regulated in the urine of women with endometriosis. CONCLUSION(S) Cytokeratin-19 may be a valuable urinary biomarker for endometriosis.

Infection of plant-parasitic nematodes by Paecilomyces lilacinus and Monacrosporium lysipagum

Studying the mode of infection of a biocontrol agent is important in order to assess its efficiency. The mode and severity of infection of nematodes by a soil saprophyte Paecilomyces lilacinus (Thom) Samson and a knob-producing nematode trapping fungus Monacrosporium lysipagum (Drechsler) Subram were studied under laboratory conditions using microscopy. Infection of stationary stages of nematodes by P. lilacinus was studied with three plant-parasitic nematodes Meloidogyne javanica (Treub) Chitwood, Heterodera avenae Wollenweber and Radopholus similis (Cobb) Thorne. Paecilomyces lilacinus infected eggs, juveniles and females of M. javanica by direct hyphal penetration. The early developed eggs were more susceptible than the eggs containing fully developed juveniles. As observed by transmission electron microscopy, fungal hypha penetrated the M. javanica female cuticle directly. Paecilomyces lilacinus also infected immature cysts of H. avenae including eggs in the cysts and the eggs of R. similis. Trapping and subsequent killing of mobile stages of nematodes by M. lysipagum were studied with the above three nematodes. In addition, plant-parasitic nematodes Pratylenchus neglectus (Rensch) Chitwood and Oteifa and Ditylenchus dipsaci (Kuhn) Filipjev were tested with M. lysipagum. This fungus was shown to infect mobile stages of all the plant-parasitic nematodes. In general, juveniles except those of P. neglectus, were more susceptible to the attack than adults.

Testing the nematophagous biological control strain Paecilomyces lilacinus 251 for paecilotoxin production

Paecilomyces lilacinus is a nematophagous fungus currently developed as a biological control agent. In order to evaluate potential toxin production, culture extract and concentrated culture supernatant of P. lilacinus strain 251 were tested against Gram-negative and Gram-positive bacteria. High-performance liquid chromatography analysis was carried out to compare the chromatograms of P. lilacinus strain 251 with the chromatogram of known paecilotoxin. It was found that the 251 strain of P. lilacinus did not produce detectable levels of paecilotoxin or other toxins with antimicrobial activity.

Fungal proteins with mannanase activity identified directly from a Congo Red stained zymogram by mass spectrometry

Secreted fungal proteins with mannanase activity were identified by mass spectrometry of bands excised from a Congo Red stained zymogram containing locust bean gum as substrate. This technique circumvents the need to locate corresponding bands on a parallel gel without substrate and provides good accuracy in targeting proteins for identification.

Rising CO2 concentration altered wheat grain proteome and flour rheological characteristics

Wheat cv. H45 was grown under ambient CO2 concentration and Free Air CO2 Enrichment (FACE; e[CO2], ∼550 μmol CO2 mol(-1)). The effect of FACE on wheat grain proteome and associated changes in the flour rheological properties was investigated. A comparative proteomic analysis was performed using 2-D-DIGE followed by MALDI/TOF-MS. Total grain protein concentration was decreased by 9% at e[CO2]. Relative abundance of three high molecular weight glutenin sub units (HMW-GS) were decreased at e[CO2]. In contrast, relative abundance of serpins Z1C and 1-Cys peroxiredoxin was increased at e[CO2]. Elevated [CO2] also decreased the bread volume (by 11%) and dough strength (by 7%) while increased mixing time. However, dough extensibility and dough stability were unchanged at elevated [CO2]. These findings suggest that e[CO2] has a major impact on gluten protein concentration which is associated lower bread quality at e[CO2].

Bead-based multiplex immuno-assays for cytokines, chemokines, growth factors and other analytes: median fluorescence intensities versus their derived absolute concentration values for statistical analysis.

Within the scientific literature, analyses of data from bead based multiplex immunoassays are based on either median fluorescence intensities (MFI) or derived absolute concentration values (ACV) but no consideration of which set of data is the most appropriate for analysis has been published. Here we look at the variance of MFI versus their ACV from the expression of 14 analytes in plasma, using 6 commercially available kits, across 177 patients, recorded at two time points and the associated analyte standards. In total 60 micro titre plates were used resulting in 4965 MFI. In doing so we develop a new background subtraction procedure that reduced by 50% the number of out-of-range values observed in our data set. Using a linear mixed-effect model, which normalizes for assay-to-assay variation, MFI produced similar significant differences than that observed using absolute concentration values. We show that subtracting analyte blanks produces 15% negative MFI resulting in uncertainty of the data being analysed. We argue for analysis of protein expression values MFI are generally a better choice than absolute concentration values. It is argued that analyte standards are not required on each plate, or not at all, in multi-plate experiments, but knowledge of the concentration curve and the range of MFI values that fall within the limits of this curve for each analyte is required. The significance of using MFI over concentration values for the life scientist means higher statistical power and lower costs.