Let’s Get Small
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.