Thermo Fisher Scientific demonstrates
superiority of Data-Dependent Decision Tree Logic

Combined intelligent ETD and CID Fragmentation Approaches for Tyrosine Phosphorylation Site Determination is available on the LTQ Orbitrap XL ETD Mass Spectrometer.

Thermo Fisher Scientific's new application note entitled "Enhancing Phosphotyrosine Proteome Coverage using a Combined ETD and CID Approach on a LTQ Orbitrap XL ETD" is available now. The application note is a result of the cooperation with the Netherlands Proteomic Centre and in particular the Biomolecular Mass Spectrometry and Proteomics Group at Utrecht University, headed by Albert Heck. It demonstrates how electron transfer dissociation (ETD) and collision induced dissociation (CID) are complementary fragmentation techniques, that when used together on the Thermo Scientific LTQ Orbitrap XL ETD mass spectrometer, provide significantly greater phosphoproteome coverage than mass spectrometry (MS) methods relying on CID alone. By using intelligent data-dependent decision tree (DDDT) logic to choose whether to perform ETD or CID in real-time, significantly more phosphopeptides can be identified, along with unambiguous determination of the phosphorylation sites.

Reversible tyrosine phosphorylation plays an important role in many cellular processes such as growth, differentiation and migration. Because aberrant tyrosine phosphorylation has been shown to affect the course of many cancers, the detection and site-specific localization of tyrosine phosphorylation are important to understanding cancer mechanisms. Low abundances (compared with non-phosphorylated peptides) and the dynamic nature of tyrosine phosphorylation make it difficult to detect. A variety of strategies, including immuno-affinity enrichment of phosphoproteins and phosphopeptide enrichment prior to MS, have been developed to overcome underrepresentation of tyrosine phosphorylation. Despite advances in these approaches, characterization of the tyrosine phosphoproteome is far from comprehensive and ongoing method development is needed.

The application note compares two approaches to identify and localize tyrosine phosphorylation in a phospho-tyrosine peptide immuno-affinity purification of pervanadate treated HeLa cells. One is the traditional approach of fragmenting every peptide by both ETD and CID. The other approach is to use DDDT logic in real time to choose the most effective dissociation technique (ETD or CID) depending on the peptide's mass-to-charge ratio (m/z) and its charge state (z).

The LTQ Orbitrap XL ETD acquires high-resolution full-scan spectra in the Orbitrap mass analyzer, and then uses the linear ion trap to perform either ETD or CID. Its superior mass resolution enables DDDT logic to use the information in the full-scan spectra to make real-time decisions about which fragmentation method to use. Data was processed with the Thermo Scientific Proteome Discoverer software, which is specifically designed to handle raw data with CID and ETD spectra. It provides search algorithms for ETD data and combines results from multiple search engines and dissociation techniques.

Compared with the traditional approach, 30% more tyrosine phosphopeptides were identified when using intelligent DDDT logic. In both approaches, a significant number of phosphopeptides were identified only by ETD or CID, demonstrating that the techniques are truly complementary. ETD is most effective for peptides with three or more charges, while CID is most effective for those doubly charged. The nearly complete fragment ion series produced from complementary ETD and CID spectra generated using real-time DDDT logic enabled unambiguous determination of the tyrosine phosphorylation sites.

For further information or to download the application note, e-mail: analyze@thermo.com or view website: www.thermoscientific.com/orbitrap  Refer to page 296

Introducing the new Diffusion Measurement package
for the Olympus FluoView FV1000 confocal microscope

Olympus' new Diffusion Measurement module for Olympus ASW 2.1 software is designed specifically for use with the Olympus FluoView FV1000 confocal microscope system for live cell imaging, this new software module provides users with the flexibility to undertake three different types of diffusion study: point fluorescence correlation spectroscopy; raster scan image correlation spectroscopy (RICS) and fluorescence recovery after photobleaching (FRAP). As a result, the Diffusion Measurement Package is ideal for a broad range of cell biology and biophysics applications.

For further information contact, Esther Ahrent, e-mail: microscopy@olympus-europa.com or view website: www.microscopy.olympus.eu  Refer to page 298   

The modular syringe-based
Droplet Starter System from Dolomite

Dolomite's, modular Syringe-based Droplet Starter System, enables users to produce more than 10,000 monodispersed droplets per second, ranging from Ø 20 to 250µm. Operating over a wide flow range, from 0.1µl/min to 10ml/min, this chemically resistant system is ideal for initial concept work and experimentation towards the development of high throughput droplet systems. Including a range of Droplet Junction Chips with different geometries, channel sizes and surface properties, the Syringe-based Droplet Starter System enables the formation of both water-in-oil and oil-in-water droplets.

This easy-to-use system is the latest addition to Dolomite's Micro Droplet System range and comprises of two independent syringes, with rotary valves, enabling fast automatic refill from up to 3 different sources, as well as an independent control of each liquid channel. As a modular system, the pump and droplet generation components are quick and simple to connect, ensuring that reliable and accurate data is obtained within a short time-span.
 
For further information on Dolomite's custom capabilities and the complete range of Micro Droplet Systems, view website: www.dolomite-microfluidics.com

Dolomite's Pressure-based Droplet Generation
System provides rapid advances in micro-droplet research

Dolomite's Pressure-based Droplet Starter System, is ideal for the initial exploration of droplet microfluidics. It provides a complete solution containing all necessary pumps, connectors and chips, enabling the immediate production of highly monodispersed droplets from Ø 20 to 150µm, with a Ø tolerance of +/- 1 %. Operating over a wide pressure range of 0 to 10bar, this Pressure-based Droplet Starter System is extremely chemically resistant and its modular design makes it easily expandable for future application requirements.

Providing a pulseless and stable liquid flow, this cost-effective modular toolkit eliminates dead volume and sample waste to effectively reduce associated reagent costs. In addition, the ability to provide a quick liquid changeover significantly increases the throughput of any application. A lockable pressure chamber and an innovative 3-way chamber lid allow simultaneous pumping of up to three liquids, making it ideal for entry-level experimentation.

Dolomite'S Pressure-based Droplet Generation System provides rapid advances in micro-droplet research.

For bespoke applications, Dolomite designs and manufactures custom  chips, tailored to meet the requirements of any laboratory.

For further information on Dolomite's custom capabilities as well as the entire range of Micro Droplet Systems and the complete portfolio of microfluidic products, including chips, connectors/interconnects, pumps, valves and flow sensors, view website: www.dolomite-microfluidics.com  Refer to next page

Thermo Fisher Scientific introduces new imaging ATR
accessory for fast and reliable FT-IR chemical imaging analyses

Thermo Fisher Scientific's new imaging attenuated total reflectance (ATR) accessory offers enhanced spatial resolution and high speed data acquisition capabilities for Fourier transform infrared (FT-IR) chemical imaging applications. The Thermo Scientific imaging ATR accessory has been designed to seamlessly and effortlessly integrate with the Thermo Scientific range of FT-IR microscopes to enable efficient, fast and reliable chemical imaging of challenging samples. This is of particular benefit to manufacturers of composite structures, paper products, pharmaceutical dosage forms, biological tissues, polymer films and coatings. The product was showcased at the JAIMA Expo 2011 in Makuhrai Messe, Japan.

ATR is a popular sampling technique for infrared spectroscopy allowing direct contact sampling of solids and liquids without further preparation. When coupled with an FT-IR microscope, ATR provides a unique capability for exploring the microscopic structure of heterogeneous samples. Single point ATR mapping, while more flexible in terms of total image size and dimensions, requires many separate contact points to build up the imaging data set. The Thermo Scientific imaging ATR accessory provides a more rapid method because it requires the sample to be compressed only once against an extended area of contact. Data collection may then proceed at the same speed as normal transmission or external reflectance experiments.

The new imaging ATR accessory greatly simplifies the task of obtaining high quality infrared spectra in cases of samples that are difficult to prepare for transmission analysis. Observable features in specimens, such as defects, particles, fibers or inks, may be effectively analyzed with spatial resolution improved by a factor of four relative to transmission measurements. The accessory quickly snaps into the sample holder feature of Thermo Scientific Nicolet iN10, Nicolet iN10 MX and Nicolet Continuµm microscopes in a matter of seconds without the need of tools. All standard microscope optics remain available, thus avoiding any concerns of alignment or down time when switching between sampling modes. Transmission, external reflection, micro-ATR and imaging-ATR capabilities are all immediately available to address the requirements of any FT-IR microscopy application.

For further information, e-mail: analyze@thermofisher.com or view website: www.thermoscientific.com/ftir  Refer to page 296