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Sep 15, 2009 (Vol. 29, No. 16)

HPLC Advances for Biomolecular Analysis

Enabling Tools and Technologies Make Cutting-Edge Research Possible

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    Researchers at Northeastern University are using MLAC-MS to examine differences in the glycan plasma proteome between women with early breast cancer and healthy controls.

    Another important pump feature, introduced at “HPLC” is force-feedback control, a mechanism that allows the pump to guarantee performance by delivering accurate and precise flow and gradients over the whole range of conditions, resulting in exceptional reproducibility.

    “It assesses the compressibility of the mobile phase in every stroke. Other pumps merely give an average. This is a unique way of compensating for the variation in the mobile phase and guaranteeing the performance of the pump,” Dr. Guazzotti said. Put simply, force feedback control involves continuous adjustment of valve timing and pumping efficiency based upon the measured compressibility of the solvents.
    One of the advantages of U-HPLC is that it reduces solvent usage, which has obvious benefits as the acetonitrile shortage continues. Switching to Thermo U-HPLC is just one option for those concerned about the acetonitrile shortage. Recycling and swapping acetonitrile are also possible, although the latter will produce changes in viscosity and, therefore, pressure of operation. The new Thermo Scientific SRS Pro Solvent System makes the former option easier by redirecting untainted mobile phase to the solvent reservoir during isocratic HPLC. It is powered directly from a USB connection, allowing plug-and-play operation in labs wanting to conserve solvent.

    Thermo Fisher Scientific also offers its Transcend™ TLX systems with Aria™ software, which allows multiplexing of two or four channels of HPLC-MS and U-HPLC-MS. A mass spectrometer is a significant investment, yet for much of the time it lies idle—because it only detects the compounds of interest during a fraction of the complete LC run. If a lab has four HPLCs and one MS, higher productivity is possible by having the HPLC systems multiplexed, so that the MS can be dedicated to the HPLC system that is eluting the compounds of interest.

    “The concept is easy,” said Dr. Guazzotti. “But the execution is difficult. We have developed a solution within Thermo Fisher Scientific that saves buying additional mass spectrometers by enabling users to increase the throughput of their existing MS.”

    A further Thermo Fisher Scientific development on the Transcend TLX system is TurboFlow™ technology, which allows for online sample preparation. Complex biological samples often need to be preprepared—for example, protein might otherwise be precipitated and phospholipids can induce ion suppression which hinders detection in the MS. There are various well-accepted ways of cleaning up the sample prior to HPLC, but they are all labor intensive and may be expensive. It would be better if the sample could just be directly injected and cleaned up automatically before entering the analytical column.

    With TurboFlow technology, the sample is introduced into a TurboFlow column and subjected to fast flow conditions that do not allow large molecules, like proteins, time to diffuse. The smaller target molecules with an affinity to the stationary phase, bind to the column. Then the flow is reversed, and the bound molecules elute and go to the analytical column, while unwanted molecules are washed away. “You see an improvement in data quality and a significant decrease in sample preparation time when you use TurboFlow technology compared to standard preparation,” explained Dr. Guazzotti.

    HPLC is also being applied in plasma proteomics. There is growing awareness that glycoproteins may exhibit different glycan patterns in cancer and in healthy tissue. As part of the NCI’s new glycobiology initiative—the Alliance of Glycobiologists—William Hancock, Ph.D., of Northeastern University, described the contribution of multiple lectin affinity chromatography (MLAC-MS) to the comparison of the glycan plasma proteome  between women with early breast cancer and healthy controls. 

    Andrew Alpert, Ph.D., president of PolyLC Inc. in Columbia, MD, presented the application of ERLIC (Electrostatic Repulsion HILIC) to the separation of phosphopeptides and sialylated glycopeptides, which would be difficult to separate with HILIC alone. ERLIC is proving particularly useful in investigating post-translational modifications of peptides in proteomic studies, Dr. Alpert said.

    Meanwhile, Albert Sickmann, Ph.D., Institute for Analytical Sciences, Dortmund, Germany, explained that a knowledge of platelet proteomics is important to better understand the role of platelet activation in hemostasis and thrombosis. But studying platelets is challenging—there are more than 100,000 analytes in a platelet sample and many protein isoforms.

    Moreover, the time course of platelet activation is hard to follow, and there are issues around reproducibility and sensitive quantitation of analyses. Dr. Sickmann and colleagues, however, have made progress on a comprehensive study of the platelet proteome, with the identification of 2,500 proteins, more than 1,000 phosphorylation sites, and more than 350 glycosylation sites. They are also using quantitative MS to identify changes in post-translational modification during platelet activation.

    Finally, Gabriella Massolini, Ph.D., of the University of Pavia, presented work on the phosphoproteome of amniotic fluid using HPLC-MS. “Amniotic fluid is a dynamic and complex sample that reflects the state of the fetus,” she said. Many biomarkers have been reported to be involved in fetal growth; IGFBP-1 (insulin-like growth factor binding protein) is one example. Phosphoproteins are a new type of biomarker; aberrant phosphorylation has been shown to be important in disease.

    However, there are various challenges in phosphoproteins analysis, arising from their heterogeneity, low stoichiometry, low abundance in the cell, and limitations in the dynamic range of analytical methods.
    Dr. Massolini’s team uses hybrid instruments, like LTQ-Orbitrap coupled with nano-LC, to pursue the analysis of phosphoproteins. They are looking at a new analytical platform with online digestions, online selective enrichment, and online LC-MS.

    The team has examined the phosphorylation state of IGFBP-1 which is only present at 0.2 mg/L. Although this represents 0.01% of the whole proteome, it is still one of the most abundant proteins. They carried out the analysis in miniature, using amniotic fluid from a single patient, with a view to using this approach for prenatal diagnosis. This setup seems to work well for acquiring the complete proteome and phosphoproteome of amniotic fluid. As with other proteomics studies, the challenge now is analysis and interpretation.

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