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With Roche s Resources Behind Him, Langen Delves Deeply into Proteomics Technology

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In a talk that illustrated the plethora of proteomics technologies available for researchers with ample resources, Roche’s proteomics director Hanno Langen shared new developments in his approach to separating and analyzing proteins last week at the IBC “Proteomics and the Proteome” conference in Geneva, Switzerland.

Langen, whose protein analysis techniques span the range of available separation techniques and include access to state-of-the-art mass spectrometers such as Bruker’s MALDI TOF-TOF instrument, said that he had recently begun implementing the MudPIT technique for identifying peptide fragments via mass spectrometry, and that in collaboration with Roche Biochemical had developed a low-salt recombinant trypsin assay for digesting proteins.

But a common theme in Langen’s presentation was his belief that 2D gel electrophoresis still presents the “best technology for separating proteins,” although he advised against excluding other more amenable protein separation techniques. The reason for his bias, he said, lies in the technique’s ability to distinguish between various isoforms of the same protein — a capability lost when a protein is digested and run through a liquid chromatograph. Furthermore, 1D and 2D gels are inherently “zoomable,” given the ability to narrow a pH gradient over a standard length IPG strip.

Needless to say, Langen’s approach at Roche relies heavily on methods for prefractionating proteins from tissue samples using standard fractionation technologies, prior to their separation. Langen said he relies on affinity purification to pull out specific classes of proteins, as well as a purification scheme using native conditions developed in collaboration with Walter Keller from the University of Basel. In addition, Langen has begun employing subcellular fractionation techniques, because tracing a protein back to its subcellular compartment can also give clues to its function.

With the help of prefractionation, Langen said he can generate 1D and 2D gels with no more than two or three proteins per spot, a resolution high enough to enable his group to identify the proteins via MALDI TOF or MALDI TOF-TOF mass spectrometry. Langen has also developed automated spot picking techniques that can pick out 800 spots from a standard 2D gel, and a 2D gel “grater” that spits out 6,000 pieces per gel for mass spectrometry analysis.

The most recent incarnation of his robotics technology uses 96 tips to spot up to 80 MALDI target plates in four hours, and Langen said that his group is in the process of moving to 384-spot MALDI plates for use with his Bruker MALDI instruments. With this technology in place, he said, his group will be able to generate 20,000 mass spectra in a day.

The increased spot density on the MALDI plates is possible because of Bruker’s AnchorChip technology, he said, which shrinks the size of the spots by drawing the liquid towards the hydrophilic center of each individual target. The downside, he added, is that shrinking the spots also concentrates impurities, such as any contaminating salts. To address this problem, Langen and his colleagues at Roche Biochemical developed a new low-salt formulation of recombinant trypsin to digest the peptides.

For low-molecular weight proteins amenable to multi-dimensional liquid chromatography, Langen said his group is in the process of setting up a system for automatically identifying peptide peaks with MudPIT, an algorithm developed by John Yates, a protein mass spectrometrist at the Scripps Research Institute in La Jolla, Calif.

Langen’s techniques for quantifying the expression of proteins also runs the gamut. For his 2D gel platform, he relies on fluorescent tags to identify up- or down-regulated proteins; likewise for his multi-dimensional chromatography platform he employs the ICAT reagent technology developed by Ruedi Aebersold of the Institute for Systems Biology. In addition, his group has developed whole cell labeling techniques using N-15.

As an example of how his multi-dimensional proteomics platform performs in practice, Langen described his work comparing living and dead mycobacteria. Using 2D gels, in combination with mass spectrometry, his group isolated a protein that seemed to protect the bacteria from being destroyed in the lysosome. In essence, he said, his group had found a drug target.

— JSM


 

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