2D GE Research Articles

Exposure to ultraviolet radiation causes proteomic changes in embryos of the purple sea urchin, Strongylocentrotus purpuratus

We performed experiments to determine how environmentally relevant ultraviolet radiation (UVR) affects protein expression during early development in the sea urchin, Strongylocentrotus purpuratus. To model the protein-mediated cell cycle response to UV-irradiation, six batches of embryos were exposed to UVR, monitored for both delays in the first mitotic division and changes in the proteome at two specific developmental time points. Embryos were exposed to or protected from artificial UVR (11.5 W/m 2) for 25 or 60 min. These levels of UVR are within the range we have measured in coastal waters between 0.5 and 2 m. Embryos treated with UVR for 60 min cleaved an average of 23.2 min (± 1.92 s.e.m.) after UV-protected embryos. Differential protein spot migration between UV-protected and UV-treated embryos was examined at 30 and 90 min post-fertilization using two-dimensional SDS-PAGE (2D GE). A total of 1306 protein spots were detected in all gels, including differences in 171 protein spots (13% of the detected proteome) in UV-treated embryos at 30 min post-fertilization and 187 spots (14%) at 90 min post-fertilization (2-way ANOVA, P = 0.03, n = 6). The majority of the proteins affected by UVR were subsequently identified using matrix assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF–TOF MS). Our results indicate UVR affects proteins from multiple cellular pathways and indicate that the mechanisms involved in UV-stress and UV-induced developmental delay in sea urchin embryos are integrated among multiple pathways for cellular stress, protein turnover and translation, signal transduction, cytoskeletal dynamics, and general metabolism.

Formation of pits during growth of Si/Ge nanostructures

Alternating deposition of Ge and Si in the step-flow growth regime using Bi acting as a surfactant can lead to a spontaneous formation of one atomic layer deep pits in the area of surface covered by Ge. During Si growth Ge atoms of the epitaxial 2D Ge layer move to Si step edges where stronger bonds with Si atoms are formed. Appropriate growth conditions can suppress or enhance the pit formation effect and consequently a new type of self-organized nanostructures can be formed.

Comparison of SYPRO Ruby and Flamingo fluorescent stains for application in proteomic research

Fluorescent dyes are widely used for the detection and quantitation of proteins separated by polyacrylamide gel electrophoresis. SYPRO Ruby is one such fluorescent dye widely used for this purpose. More recently, another fluorescent dye, Flamingo, is available for expression proteomic research. Using a standard ultraviolet (UV) transilluminator and a charge-coupled device (CCD)-based imaging system, the relative sensitivity of these two different fluorescent stains with regard to detection of protein spots separated by two-dimensional gel electrophoresis (2D–GE) and identification by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) were compared. Using mouse kidney and liver homogenates as well as Escherichia coli extract, we detected a greater number of protein spots using Flamingo compared with SYPRO Ruby. In addition, when we compared the number of matched peptides and the percentage of amino acid residues identified for 22 different protein spots of mouse kidney proteome, we observed a higher number of matched peptides and a higher percentage of amino acid residues for the majority of the proteins using Flamingo compared with SYPRO Ruby. Also, we were able to characterize a protein spot that can be detected by Flamingo only. Therefore, we recommend Flamingo over SYPRO Ruby to be used for studies on expression proteomics.

In this issue

AbstractDo rafts jump ship?pp. 4321–4328A raft, as most of us picture it, is a collection of floating material tied together to keep some kind of load afloat. A domain, on the other hand, is the area ruled by an individual, often based on inheritance. The cholesterol‐rich plasma membrane patches found on cell surfaces seem to fit the description of a domain reasonably well ‐ floating on the cell surface, they participate in cell signaling and endocytosis. And, presumably, when the host cell divides, its progeny receive a startup supply of domains (by foul means or fair). Kim et al. explored this question of distribution using synthetic glycosphingolipids (C8De and C8Lt). C8De stimulates endocytosis and rearrangement of domain proteins. Using lipid‐free methods, domains were isolated and analyzed by 2DGE, MALDI‐TOFMS and immunofluorescent microscopy. Proteins transferred to the surface after treatment were involved in ubiquitination, lipid transfer, integrin, small GTPases and several unknown functions. magnified imageFresh frozen versus formalin fixed ‐ and the winner is…pp. 4329–4339…biomedical research. The existence of a reservoir of millions of samples, many paired or containing areas of both normal and affected cells, has attracted many proteome researchers. These formalin‐fixed paraffin embedded (FFPE) samples, however, have proven very difficult to exploit. Addis et al, report here a protocol that yields full‐length protein products, suitable for 2D PAGE, Western (immuno) blots, LC‐MS/MS and MALDI‐MS, from FFPE sections. Protein extractions were performed using detergents and reducing agents and followed by TCA/acetone precipitation to remove interfering material. As many as 250 spots could be identified from FFPE sheep skeletal muscle, whereas 390 were found in fresh frozen samples. Not all 2D spots matched perfectly, but patterns were very reproducible and easily matched. magnified imagePeas porridge hot, peas porridge cold…pp. 4406–4415Agricultural applications of genetic engineering usually have regulatory requirements that GM offspring be essentially equivalent to the parental line with the exception of the target gene. This can be hard to do or hard to prove. Take the case of field peas, Mendel's favorite. To give the crop resistance to pea weevil, the α‐amylase inhibitor (α‐AI1) of the common bean was inserted into peas. Unfortunately, the engineers got more than they bargained for: the GM peas caused undesirable immunological effects in feeding tests. Upon proteomic analysis, Chen, et al., found a problem ‐ transfected genetic material included 33 genes that were over‐ or under‐expressed in five different patterns. Most of the problems were associated with Agrobacterium‐mediated events. Of the 16 proteins identified by 2D GE and MALDI‐TOF/TOF MS, many were potentially allergenic food storage proteins. A 2D gel is a cheap way to check the temperature of the soup. magnified image

Cover Picture: Proteomics 18'09

AbstractDo rafts jump ship?A raft, as most of us picture it, is a collection of floating material tied together to keep some kind of load afloat. A domain, on the other hand, is the area ruled by an individual, often based on inheritance. The cholesterol‐rich plasma membrane patches found on cell surfaces seem to fit the description of a domain reasonably well ‐ floating on the cell surface, they participate in cell signaling and endocytosis. And, presumably, when the host cell divides, its progeny receive a startup supply of domains (by foul means or fair). Kim et al. explored this question of distribution using synthetic glycosphingolipids (C8De and C8Lt). C8De stimulates endocytosis and rearrangement of domain proteins. Using lipid‐free methods, domains were isolated and analyzed by 2DGE, MALDI‐TOFMS and immunofluorescent microscopy. Proteins transferred to the surface after treatment were involved in ubiquitination, lipid transfer, integrin, small GTPases and several unknown functions.Kim, S.‐Y. et al., Proteomics 2009, 9, 4321–4328.Fresh frozen versus formalin fixed ‐ and the winner is……biomedical research. The existence of a reservoir of millions of samples, many paired or containing areas of both normal and affected cells, has attracted many proteome researchers. These formalin‐fixed paraffin embedded (FFPE) samples, however, have proven very difficult to exploit. Addis et al, report here a protocol that yields full‐length protein products, suitable for 2D PAGE, Western (immuno) blots, LC‐MS/MS and MALDI‐MS, from FFPE sections. Protein extractions were performed using detergents and reducing agents and followed by TCA/acetone precipitation to remove interfering material. As many as 250 spots could be identified from FFPE sheep skeletal muscle, whereas 390 were found in fresh frozen samples. Not all 2D spots matched perfectly, but patterns were very reproducible and easily matched.Addis, M. et al., Proteomics 2009, 9, 4329–4339.Peas porridge hot, peas porridge cold…Agricultural applications of genetic engineering usually have regulatory requirements that GM offspring be essentially equivalent to the parental line with the exception of the target gene. This can be hard to do or hard to prove. Take the case of field peas, Mendel's favorite. To give the crop resistance to pea weevil, the α‐amylase inhibitor (α‐AI1) of the common bean was inserted into peas. Unfortunately, the engineers got more than they bargained for: the GM peas caused undesirable immunological effects in feeding tests. Upon proteomic analysis, Chen, et al., found a problem ‐ transfected genetic material included 33 genes that were over‐ or under‐expressed in five different patterns. Most of the problems were associated with Agrobacterium‐mediated events. Of the 16 proteins identified by 2D GE and MALDI‐TOF/TOF MS, many were potentially allergenic food storage proteins. A 2D gel is a cheap way to check the temperature of the soup.Chen, H. et al., Proteomics 2009, 9, 4406–4415.

Nanoscale Pit Formation at 2D Ge Layers on Si: Influence of Energy and Entropy

The structural stability of two-dimensional (2D) SiGe nanostructures is studied by scanning tunneling microscopy. The formation of pits with a diameter of 2-30 nm in one atomic layer thick Ge stripes is observed. The unanticipated pit formation occurs due to an energetically driven motion of the Ge atoms out of the Ge stripe towards the Si terminated step edge followed by an entropy driven GeSi intermixing at the step edge. Using conditions where the pits coalesce results in the formation of freestanding 8 nm wide GeSi wires on Si(111).

Human K10 epithelial keratin is the most abundant protein in airborne dust of both occupied and unoccupied school rooms

Previously it was demonstrated that the levels of large particles (>2 micron) and associated bacterial cell envelope markers increase greatly on occupation in schools; it was hypothesized that the source of both was shed human skin. In the current work to test this hypothesis, room air cleaners were used to collect airborne dust (>50-100 mg) from occupied and unoccupied school rooms which was then subjected to proteomic analysis. Proteins were extracted from the dust and separated using two dimensional gel electrophoresis (2D GE). In situ digestion of protein spots with trypsin released peptides, which were subsequently analyzed by matrix assisted laser desorption/deionization, time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry (MALDI-TOF-MS-MS). In Coomassie blue stained gels, a single spot generally dominated the 2D gels; this protein was identified by tandem mass spectrometry as K10 epithelial keratin. The results experimentally confirm previous anecdotal reports that human skin is readily shed into air and suggest that increased levels of microbial markers and large particles observed in occupied rooms are also derived from skin.

Strain and electronic structure of Ge nanoislands on Si(111)-7×7surface

To study the relation between the strain and the electronic structure in two-dimensional (2D) Ge nanoislands on the Si(111)-$7\ifmmode\times\else\texttimes\fi{}7$ surface, we calculated the relation based on the density functional theory and measured with the angle resolved ultraviolet photoelectron spectroscopy (ARUPS) and the scanning tunneling microscopy (STM). In the calculation, the dangling-bond state at the strained adatom on the 2D Ge island (${S}_{Ge}^{R}$ state) shifts to lower energy at the rate of $\ensuremath{-}8.2\phantom{\rule{0.3em}{0ex}}\mathrm{eV}∕\mathrm{nm}$ with the height of the adatoms. The ARUPS spectrum shows a characteristic peak corresponding to the ${S}_{Ge}^{R}$ state, whose energy depends on the size of the Ge nanoisland. The state appears at $\ensuremath{-}0.7\phantom{\rule{0.3em}{0ex}}\mathrm{eV}$ below the Fermi level for the small Ge islands (3.8--5 nm) and at $\ensuremath{-}0.6\phantom{\rule{0.3em}{0ex}}\mathrm{eV}$ for the large Ge islands. The height of the Ge islands estimated from the energy difference is consistent with a result of the STM measurement. The strain of Ge islands can be estimated from the electronic structure.

Organellar Proteomics

The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.

Efficacy of Contrast Medium Use for Neuroimaging at 3.0 T

As inversion-recovery (IR) technique improves T1 contrast at high field strength, signal enhancement by T1-shortening contrast media may be affected. To clarify the different enhancement properties at 3.0 T, the authors compared T1-weighted sequences. Twelve contrast-enhancing lesions were investigated by spin-echo (SE), inversion recovery fast spin-echo (IR-FSE), two-dimensional gradient-echo (2D GE), and magnetization-prepared three-dimensional gradient-echo (3D GE) sequences and evaluated by comparing signal-intensity enhancements within the lesions. In addition, signal-to-noise-ratios (SNR) and contrast-to-noise-ratios (CNR) were measured. On average, signal enhancement of the lesions amounted to 60% for SE, 57% for IR-FSE, 32% for 2D GE, and 35% for 3D GE images. CNR of gray matter versus white matter was significantly higher for IR SE and GE imaging than for genuine SE and 2D GE acquisitions (Wilcoxon test), while 2D GE imaging alone had an excellent SNR. As IR-FSE images provide an excellent CNR for gray and white matter in the brain and contrast enhancement performs almost similarly well compared with SE imaging, this technique appears to be well suited for T1-weighted neuroimaging without and with contrast enhancement at 3.0 T. However, the inherent blurring of the IR-FSE can lead to poor performance for very small lesions.

Mechanical distinction and manipulation of atoms based on noncontact atomic force microscopy

Using noncontact atomic force microscopy (NC-AFM), we have succeeded in imaging two-dimensional (2D) Ge islands as well as Ge atom clusters on Si(1 1 1)-(7 × 7) surface with atomic resolution. We have investigated the tip-sample distance dependence of NC-AFM images of oxygen adsorbed Si(1 1 1)-(7 × 7) surface, and achieved the chemical distinction between oxygen and Si atom species. Besides, using a soft nanoindentation based on the NC-AFM method for mechanically manipulating Si adatoms of the Si(1 1 1)-(7 × 7) surface, we have achieved atom removal, i.e., vertical manipulation, at tip and sample temperature of 78 K as well as at room temperature (RT). In addition, as an application of this soft nanoindentation method, we have produced the lateral displacement of Si adatoms towards metastable positions in the half unit cell of the Si(1 1 1)-(7 × 7) reconstruction at 78 K.

Nucleation and Growth of Ge at Pb/Si(111) Surfaces: Reaction-Limited Aggregation

The nucleation and growth of two-dimensional (2D) Ge islands at Pb-layer-covered Si(111) surfaces is studied using scanning tunneling microscopy (STM). Ge adatoms are highly mobile on the Pb layer. There is a threshold deposition coverage for nucleation of Ge islands to occur. Above it, the observed island density increases rapidly but the average island size decreases rapidly first and then increases slowly again. Our results indicate that the nucleation and growth processes are hindered by high energy barriers required for Ge clusters to exchange with the Pb atoms. A compact-to-fractal island shape transition is observed as the deposition flux is lowered, as the temperature is raised, or at a low Ge coverage, which contradicts previous theoretical predictions and experimental findings. Our results may shed much needed light on the fundamental mechanism in surfactant-mediated epitaxy (SME).

Direct Observation of Reaction-Limited Aggregation on Semiconductor Surfaces

We report a discovery that the nucleation and growth of two-dimensional (2D) Ge islands at a Pb layer covered Si(111) surface are reaction limited. Using scanning tunneling microscopy, a compact-to-fractal island shape transition is observed as the deposition flux is lowered, the temperature is raised, or at a low Ge coverage. This behavior is completely opposite to what was predicted from those theories based on diffusion-limited aggregation and previous experimental observations. Energy barriers are found to exist for the nucleation and growth of Ge islands, indicating that their growth behavior is exchange-reaction rate limited.

Instability of 2D Ge layer near the transition to 3D islands on Si (111)

The formation of a stable surface morphology of Ge on Si(111) at coverages close to the transition from two-dimensional (2D) to threedimensional (3D) growth was studied using scanning reflection electron microscopy and energy dispersive X-ray spectroscopy. Regions of the stable surface morphologies of 2D layers and 3D islands are described by a phase diagram as a function of coverage and temperature. Temperatures were determined at which the unstable 2D Ge layer at coverages between 1.5 and 2.3 BL does not transform into islands longer than 10 min. It was found that irradiation by focused e-beam creates points in the unstable 2D layer where 3D islands appear after annealing. These 3D islands probably nucleate on structural defects introduced by the irradiation. This shows that the instability of the 2D layer can be used for controllable nucleation of islands.

Magnetic resonance imaging in hemophilic children: value of Gradient Echo and contrast-enhanced imaging

The purpose of this study was to determine the value of Gradient Echo imaging for the evaluation of cartilage (3D fatsat) and blood products (2D Hemoflash), and the use of contrast enhanced SE imaging for the evaluation of synovial changes, in comparison to the clinical evaluation of children with hemophilia A. We investigated 21 joints in 16 patients with evidence of hemophilia A (mean age 11.3 ± 2.1 years). In all patients, clinical examination, plain film radiographs, and MR evaluation were performed magnetic resonance imaging (MRI) was performed by using sagittal T 1 SE and T 2 SE images, as well as 3D fatsat GE and 2D GE images. Axial and sagittal T 1 weighted SE images were obtained before and after contrast application. Findings from the clinical examination and MR imaging, regarding the evaluation of blood, synovia, and cartilage were compared. Clinical examination revealed evidence of a bleeding episode in 12 joints (57.1%), whereas MRI revealed evidence of blood or blood products in 15 joints (71.4%). Clinical investigations, including bleeding scores, pain scores, and physical examination scores did not correlate with MR findings. Due to the MR findings in 6 of 16 patients, therapeutic management was changed from on demand to prophylactic therapy. MR imaging with gradient echo and contrast-enhanced sequences is more sensitive than clinical examination for the detection of blood products in children with hemophilia. Its ability to demonstrate potentially early stages of cartilage or synovial alterations might assist in therapy planning. Clinical scores might underestimate effects of hemophilia.

Exchange-Barrier Effects on Nucleation and Growth of Surfactant-Mediated Epitaxy

The initial stage of nucleation and growth of two-dimensional (2D) Ge islands on Pb covered Si(111) surfaces at room temperature is studied using scanning tunneling microscopy. The Pb overlayer is found to greatly enhance surface diffusion of deposited Ge adatoms. There is a critical deposition time above which the observed island density increases sharply while the average island size decreases. We believe nucleation as well as growth is initially hindered by the high energy barriers for Ge clusters to exchange with Pb atoms until Ge clusters reach a critical size. A mechanism is proposed to explain the observed phenomena.

Interface Morphology of Gen / Si Quantum Wells Studied with Raman Spectroscopy and High Resolution X-Ray Diffraction

We report Raman scattering and high-resolution X-ray diffraction measurements performed on a series of MBE-grown Si / Gen / Si quantum wells with n varying from 3 to 6. Our results are consistent with a gradual evolution of the Si / Ge interface which starts with Ge segregation and formation of terraces for low Ge coverage, to smooth 2D Ge layers, bounded by interfacial alloy layers, for coverages superior to 4M Ls.

MRI and CT of tumors of the pharynx: comparison of the two imaging procedures including fast and ultrafast MR sequences

The aim of this prospective study was to compare the diagnostic value of computed tomography (CT) and magnetic resonance imaging (MRI) in the evaluation of tumors of the nasopharynx, oropharynx and hypopharynx. For this reason, we performed CT and MRI in a total of 473 patients with neoplasms of the nasopharynx [116], oropharynx [282] and hypopharynx [39]. Furthermore, the MR imaging protocol had to be optimized including 2D and 3D gradient echo sequences. We compared the use of conventional and fast spin echo and 3D gradient echo sequences for the evaluation of tumors of the nasopharynx and the skull base, and we evaluated 2D fast and ultrafast MR sequences for the detection and delineation of tumors of the oropharynx and hypopharynx. MRI yielded better results than CT in the detection of pharyngeal neoplasms. The differentiation of tumor and inflammatory tissue was best achieved using MRI. Contrast-enhanced 3D GE sequences improved the diagnostic value of MRI of the nasopharynx. Fast and ultrafast 2D MR sequences increased the contrast between orofacial tumors and surrounding tissues. Functional MR images (phonation studies, Valsalva's maneuver) were attainable using ultrafast sequences thus enhancing the diagnostic value of MRI in the case of tumors of the hypopharynx. T2-weighted turbo (fast) spin echo sequences with relatively short acquisition times proved to be superior to conventional T2-weighted spin echo sequences. In conclusion, MRI is the imaging modality of choice in the evaluation of pharyngeal neoplasms. Conventional T2-weighted SE should be substituted by fast T2-weighted SE sequences. 3D GE sequences should be integrated in the MR imaging protocol of the nasopharynx. Fast and ultrafast 2D GE sequences should be applied in the evaluation of tumors of the oropharynx and hypopharynx.

MRI of orofacial tumors and paragangliomas with 2D GE sequences: indications and optimal sequence parameters

The aim of this paper is to determine to what extent and in which cases 2D gradient echo (2D GE) sequences can be applied alternatively or additionally to spin echo (SE) sequences for improved diagnostic evaluation. Imaging with SE sequences is the most frequently used MR technique in the assessment of ear, nose and throat (ENT) tumors. In literature there are only a few reports on the contrast behaviour of 2D GE sequences using different sequence parameters and their application in ENT tumors. This paper set out to establish the most suitable sequence type and sequence parameters. Measurements were performed with a Magnetom SP 63 MR system (Siemens) with a field strength of 1.5 T, using head and Helmholtz coils. One-hundred twenty-eight volunteers and 369 patients were examined with 2D GE sequences. In order to find the best MR technique for the examination of orofacial tumors and paragangliomas, FLASH, FISP and PSIF sequences with different sequence parameters (TR, TE, flip angle, bandwidth) were applied. The results of these examinations confirmed the superiority of 2D GE sequences over SE sequences. In conclusion, contrast enhanced or unenhanced T1 weighted SE sequences should be replaced by 2D GE FLASH 70° in the examination of orofacial tumors. In the case of paragangliomas of the jugular bulb and the parapharyngeal space T1- and T2-weighted SE sequences should be replaced by 2D GE FLASH 40°.

Fabrication of patterned Gex/Si1−x/Si layers by pulsed laser induced epitaxy

Selective growth of GexSi1−x on Si is demonstrated for the first time using a pulsed laser induced epitaxy technique, combined with either standard oxide or liftoff patterning processes. Two different dimensions of Ge0.12Si0.88/Si lateral wells are formed, 3.5 μm wide by 1700 Å deep, and 6 μm wide by 1300 Å deep. High-resolution transmission electron microscopy, combined with energy-dispersive x-ray imaging, reveals a well-defined two-dimensional (2D) Ge redistribution profile with no significant line or surface defects observed. The 2D Ge well distribution profiles, governed by heat and mass transport during the laser processing, are discussed.