Leading the Way in Life Science Technologies

GEN Exclusives

More »

Feature Articles

More »
May 15, 2012 (Vol. 32, No. 10)

Maximizing Mass Spec's Potential

  • Click Image To Enlarge +
    View into the inlet of a flight tube of a triple quadrupole mass spectrometer. The use of mass spec in life science research is exploding. [Jens Goepfert/ Shutterstock Images]

    No longer confined to the realm of specialized laboratories, the use of mass spectrometry (MS) has exploded of late. Researchers from a variety of disciplines contribute to making the analysis of ever smaller samples of macromolecules faster, easier, more streamlined, less error-prone, and less paradoxical.

    At the “International Symposium on Microscale Bioseparations and Analysis” held earlier this year, scientists discussed their research into various tools and techniques used in combination with MS.

    Hartmut Schlüter, Ph.D., professor at the University Medical Center Hamburg-Eppendorf, concentrates on mass spectrometric proteomics—for example, detecting, purifying, and identifying unknown proteases for which the catalytic reaction is known. He described a special form of two-dimensional displacement chromatography

    using the Agilent Technologies HPLC-Chip, a miniaturized system designed to separate small amounts of peptides—down to femtomoles to attomoles—for analysis by mass spectrometry.

    “In collaboration with Ulrich Tallarek from the University of Marburg and Agilent Technologies, we are trying to improve the HPLC-Chip technique,” he explained.

    Driven by pulsed injections of a displacer molecule, samples pass through a cation exchange (CEX) column—the first dimension—on the way to the chip, which itself consists of two columns. The first is a reverse-phase trapping column where tryptic peptides are enriched and desalted. A valve—also present on the HPLC-Chip—is then switched for the peptides to be eluted to and separated by a second reverse-phase column. From there, fractions are ejected out the integrated nanospray emitter into the mass spectrometer via an ionization chamber.

    Peptides compete with each other for binding to the CEX stationary phase during loading, with those of the highest affinity remaining nearest the column inlet. Yet instead of using a salt gradient to elute peptides from this first dimension, Dr. Schlüter’s team uses spermine to displace the peptides from the column. Spermine competes with the peptides for binding to the stationary phase, so rather than diluting and potentially mixing the fractions, they are focused and concentrated.

    Displacement chromatography faces at least two challenges. First, it’s critical to adjust the amount of sample to the binding capacity of that sample to the column—yet determining what that binding capacity is can itself waste potentially precious sample. Second, continuous flow of displacer—typical for one-dimensional or off-line separations—in online chromatography doesn’t allow time for samples to separate on the second, reverse-phase, column.

    They were able to overcome these challenges by using repeated pulsed injections of sample, slightly overloading the column. Using LC-MS to monitor everything that passed through the CEX column following each injection, they determined that the column was fully loaded for the first low affinity peptides eluted.

    Similarly, using small, pulsed injections of spermine and monitoring the LC-MS chromatograms after each allows for a physical separation of the eluted fractions and optimization on the fly. There is a significant gain in resolution over that of a salt step gradient typically applied, but it comes with a price, Dr. Schlüter noted, in that pulsed displacement chromatography is a far more time-consuming endeavor.

  • Let’s Get Small

    Click Image To Enlarge +
    Researchers at the University of North Carolina are working on integrating a microfluidic capillary electrophoresis platform with electrospray ionization.

    Charged molecules can also be separated by electrophoresis. This can be done about 100 times faster on microfabricated structures than in capillary formats, pointed out J. Michael Ramsey, Ph.D., professor of biomedical engineering at the University of North Carolina at Chapel Hill. Case in point: platforms such as the Agilent 2100 Bioanalyzer. “But it has been limited to using fluorescent tags basically to identify biopolymers—DNA and proteins.” Electrophoretic mobility by itself essentially indicates little more than a relative measure of size.

    Dr. Ramsey, who was the scientific founder of Caliper Technologies (now part of PerkinElmer), has been working on integrating a microfluidic capillary electrophoresis (CE) platform with electrospray (ES) ionization.

    “So you can do these same types of high-performance separations that now get the biomolecules out into the gas phase so you can do MS on them,” he said, and allow chemical information such as molecular weight and size:charge ratio to be extracted. “In proteomics types of applications, that allows you to do anything from identifying amino acids and peptides to determining post-translational modifications on intact proteins, or even sequencing proteins if you can do tandem MS.”

    Typically a CE experiment would involve using a silica capillary for the electrophoresis experiment and a separate pulled capillary to be used as a nanoelectrospray orifice, with the two combined through a liquid junction. The latter is what allows a potential to be applied at the distal end of the capillary to establish an electrophoretic field.

    Spraying from a sharp tip allows the electric field lines to be concentrated, generating a higher electric field. Dr. Ramsey’s group has implemented the electrospray capability right onto the chip. There is a “feature in the corner of the chip where two channels meet to essentially form a liquid junction, and then a channel leading to the corner of the chip serves as an ES emitter,” he explained.

    Besides pulling biomolecules along the capillaries and into an ionization chamber, electrical potential is also used to control dynamic valves—which determine whether sample flows down into the separation channel or gets shunted to a waste reservoir, replacing a more traditional mechanical mechanism.

    Building on this work, Dr. Ramsey also demonstrated a dual ES emitter with two separate emitters on a single-chip structure. These can be electronically modulated on and off, allowing them to achieve the same sort of result as having two separate orifices controlled by a manual shudder—typically used to achieve higher mass accuracy.

    In mass spectrometry-based proteomics, sample preparation can be critical. There is a lot of room for human error in extraction, enrichment, filtering, desalting, dialysis, and other manipulations, and discrepancies can compound and propagate throughout the process.

    “If you have a lot of steps that are discontinuous and involve a lot of sample treatment in between, you’re going to lose sample and you’re going to introduce error throughout,” said Ziad El Rassi, Ph.D., chemistry professor at Oklahoma State University. “Then when you do MS comparative assessment, there is always some question whether the samples went through the same sample loss, so your comparison may not be accurate.”

    Unlike DNA, there is nothing against which to “normalize” the amount of a protein in a given sample. All that can be done is compare samples to one another, and for this to have meaning samples need to be treated in the same way.

    By integrating six micropreparative scale HPLC columns into a cascade-like operation via multiple switching HPLC valves, Dr. El Rassi and his colleagues are able to keep the sample moving “from one step to another without collecting, without sample treatments, without anything,” he explained. The platform allows samples to be extracted, captured, concentrated, and fractionated using a variety of techniques including affinity- and reverse-phase chromatography, all while remaining in the mobile liquid phase.

    In the final chromatographic step, the sample is collected in an organic-rich mobile phase, followed by evaporating the volatile solvent, and subsequently tryptically digested. “That’s the only step required from inlet to outlet to make the sample ready for MS,” he noted.

    Each run can process about 10 µL of serum, and with several runs per day, the platform allows about 100–200 µL to be processed per day, “which is a lot for LC-MS/MS,” Dr. El Rassi claimed. He demonstrated its use in the comparative analysis of the fucosylated sub-glycoproteomes of disease-free and breast cancer sera.

Related content

Be sure to take the GEN Poll

Cancer vs. Zika: What Worries You Most?

While Zika continues to garner a lot of news coverage, a Mayo Clinic survey reveals that Americans believe the country’s most significant healthcare challenge is cancer. Compared to other diseases, does the possibility of developing cancer worry you the most?

More »