Innovation in LC/MS
Beckman Coulter’s ProteomeLab PF 2D (Proteome Fractionation in 2 Dimensions) System fractionates a proteome in the first dimension by pH using chromatofocusing, and then automatically injects each fraction into the second dimension for separation by hydrophobicity.
The system generates liquid fractions of the proteome as well as a pH/hydrophobicity 2-D map that identifies regions of differential protein expression. With up to a 5-mg mass load, the system allows the isolation of low-abundant proteins at levels that can readily be identified by mass spectrometry, according to the company.
Over the past five years, Dominic Gostick, Ph.D., director of proteomics for the ToF-MS product line at Applied Biosystems (www.appliedbiosystems.com), has observed a trend away from traditional 2-D gel electrophoresis and a protein identification-based workflow to an LC/MS workflow and an emphasis on identification and quantitation. The introduction of istopic and isobaric labeling reagents has enabled relative quantitation of proteins and multiplexing at the level of the mass spectrometer.
The move from a 2-D gel world to the world of LC and mass spectrometry has enabled people to better deal with the complexity in their proteomics samples by having a liquid chromatography step upfront, says Dr. Gostick.
At the end of 2005, Applied Biosystems introduced Tempo LC, a range of low-flow chromatography systems designed as front-ends for either ESI or MALDI MS. In January, the company launched the QSTAR Elite LC/MS/MS system, designed to offer improved resolution, dynamic range, and speed of MS/MS for proteomics applications. The biggest challenge today is dynamic range, says Dr. Gostick. Both 1-D and multi-dimensional chromatography are enabling us to go deeper into the proteome.
Eksigent Technologies’ nano-scale LC systems designed for nanospray mass spectrometry incorporate microfluidic pumping technology to deliver precise, low-flow rates down to 100 nL/min gradients, without the need for flow splitting. The Nano LC-1D and 2-D systems integrate with MS instrumentation from major manufacturers through Eksigent’s LC Control software to enable LC/MS identification of proteins and peptides in proteomics applications such as biomarker discovery.
The 2-D system can perform online multidimensional separations, typically with an ion exchange column in the first dimension and a reverse-phase column in the second dimension. The company introduced the Nano LC-1D Plus in 2005, which includes a third pump for rapid sample loading and washing.
Bailey identifies five main areas in which HPLC-based sample-prep strategies will evolve. These include new affinity-capture technologies; continued integration of protein separations with LC/MS hardware and software; increased recovery of protein samples during sample prep and separations; continued improvement of multidimensional separation techniques to enable new workflows for biomarker discovery; and greater emphasis on whole protein capture and separations to preserve and maintain post-translational modifications information.
Bailey emphasizes industry demands for improved reproducibility, increased sample-loading capacity, high protein recovery workflows, better selectivity, reduced sample-preparation time, and increased automation.
Agilent’s proteomics separation suite consists of four main product families: the multiple affinity removal system, macroporous reverse-phase (mRP) columns, HPLC chip products, and the soon-to-be-released OFFGEL Fractionator.
GE Healthcare’s Ettan MDLC and Ettan nanoLC can be used in combination with the company’s new Decyder MS differential analysis software, which allows users to import retention times/chromatograms with the mass-to-charge data from the mass spec and analyze the LC/MS data as a 2-D plot or a 3-D intensity plot. In this way you can visualize anomalies in the data that couldn’t as easily be seen on the mass spectra or chromatograms, explains Gruia-Gray. These findings can be used to evaluate the quality and reproducibility of sample-prep procedures.
You can take DeCyder MS one step further, adds Gruia-Gray, and do the equivalent of DIGE, comparing samples, doing differential analysis, and doing relative abundance comparisons.
Waters’ nanoACQUITY Ultra Performance LC (UPLC) system performs capillary to nano-scale separations at flow ranges of nL/min up to µL/min in 75? micron diameter columns. The nanoACQUITY system does not require flow splitting, an advantage for customers, notes Gebler, because as the column is aging and they are putting on different kinds of samples, they don’t have to worry about the flow characteristics of the column changing. The nanoACQUITY also offers online MDLC capability.
Other Waters products with applications in proteomic sample prep include RapiGest SF reagent, BioSuite HPLC columns, Protein-Pak columns, the Sep-Pak line of SPE products, and the NanoEase nano, capillary, and trapping columns.
Thermo’s newest entry in the LC market is its line of Hypersil Gold columns. These 1.9-micron particle chromatography columns offer constant efficiency over a large flow range, says Biringer. The company’s protein separation and analysis product line includes the Finnigan LTQ, OrbiTrap, and FT hybrid mass spectrometer. In 2006, the company introduced Electron Transfer Dissociation MS as an upgrade for the LTQan ion fragmentation technology that dissociates peptides to generate sequence information.