Highlights From Joint Congress 2011: One Era Ends in Disappointment and Another Begins*

In an opening plenary, Prof. Barry Karger of the Barnett Institute of Chemical and Biological Analysis (Boston, MA) concisely defined analytical chemistry as “The science of generating relevant information by means of chemical and biological measurements.” Much of the Joint Congress 2011 focused on doing this with higher speed, resolution, and reliability. Frequently, subdisciplines in science emerge in parallel silos using similar technology, usually with different ontologies and little cross-talk. The market-driving applications vary, but the core technical principles span the applications. Leaders at CASSS (Emeryville, CA) recognized the need and opportunity to improve communication by creating a forum designed to improve technical cross-talk between scientists in adjacent silos. Thus, CASSS organized the Joint Congress 2011, held May 1–5 in San Diego, CA. The technical program consisted of three parallel tracks organized by three different scientific committees focused on Capillary Separations (35th International Symposium on Capillary Chromatography), Microscale Separations (26th International Symposium on MicroScale Bioseparations), and Comprehensive GC (8th GC×GC Symposium).

Correcting the human genome

The opening plenary started things off on a critical note. Prof. David C. Schwartz (University of Wisconsin, Madison) is correcting the numerous errors in genomes sequenced by shotgun technology by sequencing linear DNA using capture genome hybridization (CGH). Individual strands of DNA are stretched into a line on the surface of a microscope slide and cut with restriction enzymes into specific sections. Since the connectivity is established and confirmed by the site specificity of the restriction enzymes, assembling the genome is much more accurate. This technique catches duplicates, insertions, and inversions, all of which are common. The output is a series of bars that read like a bar code.

For comparison, bar codes for individuals are aligned. This reveals similarities and differences in sequence. Capture genome hybridization for humans and chimps shows that humans have 510 deletions. Generally these are for proteins associated with mental development. Another study compared wild type and cancerous breast tissue. Prof. Schwartz found that the genes from cancerous disuse had been randomized with long sequences exchanged between the 20 genes. So 10 years after the human genome was hailed as completed, we are slowly getting a much more accurate structure. Plus, we now have tools to avoid ambiguities associated with shotgun sequencing. With a correct genome to study, perhaps it will be more useful. However, it is doubtful that genomics will deliver anything close to the expectations of 5–15 years ago.

Serum biomarkers

After 10 years and billions of dollars, scientists are recognizing that serum biomarkers are in danger of being broadly and charitably classed as hype. The problem is that the concentration range of relevant analytes spans 1011 or more, which is about 107 times the dynamic range of the best detectors (mass spectrometers). In one of the most memorable lectures of all time, Prof. Giorgio Righetti (Politecnico di Milano, Italy) focused on the hopeless inadequacies associated with current enrichment techniques, including depletions. He concluded that affinity-based depletions fail to deliver relevant information on biomarkers for serum samples. Further, Prof. Righetti anticipated the question, “What technology should we use now to win fame and fortune for biomarkers?” His advice was to take any available funds to a casino and bet them. Such refreshing honesty! I know that all the audience, except one, felt relieved that they did not have to follow Prof. Righetti at the podium.

In the next lecture, Dr. Andrei Drabovich (Mt. Sinai Hospital, Toronto, Canada) showed that the search for biomarkers can succeed if one starts with cleaner and more concentrated samples. Blood collects the waste from all organs and hence is too dilute. Other fluids in the body, including seminal fluid in males, are more tractable sources. His specific goal was differential diagnosis of azoospermia in infertile males by looking for biomarkers associated with blockage or other problems. Using nanospray electrospray ionization (ESI) with selected reaction monitoring (SRM), 18 proteins were validated in several dozen clinical samples. From this, they developed a smaller panel of discriminating biomarkers with near absolute specificity and sensitivity. This obviates the need for testicular biopsy, which is a significant benefit to patients. So the take-home message is to focus on simple systems, which are more compatible with today’s tools.

For similar reasons, Prof. Karger focused on tissue samples. Today, the Barnett Institute has two major thrusts: translational medicine and translational regulatory science. Years ago, he recognized the problems with serum proteins above. Tissue was selected because it is more complex and biologically relevant than cell culture, since the structural features are potentially important. Cells are isolated by laser dissection capture. Currently, he needs about 10,000 cells, but hopes to soon reduce the sample to 1000.

The proteins are measured with bottom-up peptide sequencing using LC-MS. The data are displayed according to the spectral index that Prof. Karger reported on in 2008. When one compares cancerous and normal cells, the spectral index easily groups the proteins that are elevated or suppressed in normal or cancer tissue.

The next question is: Are the correlations meaningful or just flukes? After all, biological systems have large populations, so random nonsensical correlations are probable. Thus, even at the 99% confidence level, 121 proteins were significantly different. Using network analysis, these produced two suggested networks, one of which appears to make sense and may have some therapeutic utility in the clinic.

The move down to single cells was reported in a poster from Lukas Galla and colleagues at Biefeld University (Germany). They used laser-induced fluorescence (λex = 266 nm) to detect a green fluorescent protein (GFP)-labeled protein (γ-PKC [protein kinase c]) from a single cell of the insect Spodoptera frugiperda. The cell was lysed with a 2500-V pulse at the head of a 3-cm-long CE separation capillary. The S/N was more than 10.

GC×GC

As the sun sets on the serum protein era, we have daybreak over the silo of GC×GC (aka comprehensive GC and multidimensional GC). Although multidimensional LC has been used for about 20 years, GC×GC has languished for nearly 50. The first report for GC was by M.C. Simmons and L.R. Snyder in 1958.1 Until very recently, GC×GC appeared to be a curiosity looking for a compelling application. Analysis of petroleum and petroleum streams produced pretty and inspiring chromatograms, but little more. The GC×GC track of the Joint Congress was driven by applications including:

  • Assay of edible oils for adulterants
  • Screening of food supplements for active and illicit analytes
  • Sports doping
  • Assay of sulfur compounds in coal and heavy petroleum
  • Assay of essential oils in fragrances
  • Assay of enantiomeric purity.

Instrumentation has improved sufficiently that it is now easier and quicker to use GC×GC for a difficult separation than to optimize a long run for a difficult pair. Plus, the ability to concentrate analytes during the run improves method detection sensitivity. For example, a poster presented by Brian Barnes (Seton Hall University, South Orange, NJ) reports development of a solid-phase micro extraction (SPME)-GC×GC time-of-flight (ToF)-MS assay for cocaine and salvinorin A of 41 and 33 ppb in urine. This is significantly more sensitive than other assays. It was pointed out that GC×GC ToF-MS is particularly powerful for assay of drug metabolites, even fast metabolizers.

Two lectures focused on GC×GC technology that can rapidly increase separation speed. Prof. R.J. Simonson (Sandia National Laboratories, Albuquerque, NM) needed to miniaturize a GC and get a peak capacity of 100 peaks per second for military applications. The goal was to make a portable instrument that could assay airborne toxins in 4 sec at the 1-ppt level. This requires generation of peak capacity of 50 peaks per second using air as the sample stream and carrier gas. The peak capacity is only possible with GC×GC. They finally used a micromachined column with a 90 cm length in the first dimension and a 30-cm-long wax column machined into a 1-cm2 wafer. Injection and flow modulation are controlled by micromachined valves.

3-D GC

If GC×GC is powerful, what about adding even another GC stage? After all, in LC, up to six stages have been commercialized. For triple-stage GC, Prof. Robert E. Synovec (University of Washington, Seattle) estimated that the second stage of the GC should have a 4-sec run time, and the third stage would need to be complete in about 200 msec. He started experimental work with the third stage and found that commercial big-box instruments were not fast enough to be useful.

This led to a critical review of the sorry state-of-the-art in big-box GCs. Prof. Synovec focused on extracolumn band broadening for low Kʹ peaks. An unretained peak (Kʹ = 0.0) could be as narrow as 5 msec with no column, but the minimum that could be measured with a big box was about 2 sec, even with flame ionization detection (FID). When a column is added (40 m × 180 μm), the peak width should be 400 msec, but the measurement is 3 sec. While the extracolumn band broadening may not be objectionable in some applications, in GC×GC it really slows things down. In one example, the column set had a peak capacity of 1000 peaks per min, which is about 10 times more than is commonly achieved with GC×GC.


Comments