When thinking “high-throughput” in the life sciences, technologies like microarrays and microfluidic chips generally come to mind—not mass spectrometry. But the quest for high-throughput approaches in biomarker and drug discovery over the last decade has led to rapid development of new proteomics methods and instruments that allow users to screen thousands of samples in a short period of time. As it turns out, mass spectrometry is ideal for this purpose. “You’re not going for the identification or sequence identity of every molecule in your sample when you’re doing high-throughput, you’re taking a broad look to pick out differences in masses—which is what the mass spec does well—and then zeroing in on those particular masses of interest,” says Mary Lopez, strategic collaborations leader, PerkinElmer Life and Analytical Sciences.
One technological improvement that has moved high-throughput mass spectrometry forward is the development of rapid, high-resolution chromatography systems. For instance, Waters’ ACQUITY UPLC system, which combines sub-2 micron small particle column chemistry with ultra high-pressure fluidics, offers up to nine-fold reduction in run times without sacrificing resolution, according to company literature. “Now where an analysis might take 15 minutes [with conventional HPLC], I can do it in three or four. I get the same quality, if not better quality with the UPLC than I did with the HPLC,” says John Shockcor, business development manager for metabolic profiling, Waters Corp.
Though unique to the market when introduced in 2004, the UPLC system now faces competition; Agilent recently premiered its 1200 Series Rapid Resolution, which likewise employs sub-2 micron column particle chemistry and high flow rates and pressures. According to the company, the technology can reduce run times up to 80%. Additionally, the system is modular and can be used for both traditional HPLC and rapid applications. “In a single pump design, you can span the traditional separations up through the rapid resolution and have broad flow rates available to you,” says John Fjeldsted, R&D director, LC/MS, proteomics, and metabolomics, Agilent Technologies. Shockcor notes that the use of UPLC does not preclude scientists from using traditional HPLC for specialty applications like chiral and carbohydrate analysis.
With the development of ultrafast liquid chromatography comes a new challenge; the peaks coming off these new systems are significantly more dense and narrow than those coming off a traditional HPLC system. Thus fast-scanning mass spectrometers are needed to optimize peak definition. “The key to really taking advantage of UPLC with your mass spec is that it must be able to scan quickly enough to fully digitize the peaks,” notes Shockcor. Waters’ high-end Premier family of mass spectrometers and less expensive ACQUITY SQ and TQ mass spectrometer detectors scan up to 20 spectra per second. Likewise, Agilent’s new 6000 series LC/MS instruments, designed to work with the 1200 Series Rapid Resolution system, offer fast spectral acquisition times as well; Fjeldsted compares the new 6510 Q-TOF mass spectrometer to a high-speed camera. “It has mass accuracy approaching what fourier transform instruments offer, has the attomolar sensitivity that ion trap instruments traditionally have, and has a speed factor that neither have been able to achieve,” he says.
Despite advancements in chromatography and instrumentation, Shockcor notes that sample preparation remains a bottleneck in high-throughput proteomics, particularly in the analysis of serum and plasma for biomarker discovery. “[With] plasma, you may have to protein precipitate and filter and centrifuge and do all sorts of manipulations prior to the actual analysis, and a lot of times these things can be the rate-determining step,” he notes. Serum and plasma samples are particularly tricky because they include high-abundance carrier proteins that circulate in the blood and can hinder identification of low-abundance biomarkers. A number of kits are available to reduce sample complexity by removing such proteins, including Calbiochem’s ProteoExtract protein removal kits (which remove serum albumin and IgG), Agilent’s Multiple Affinity Removal System (which depletes the seven most abundant serum proteins), and Sigma-Aldrich’s ProteoPrep 20 Plasma Immunodepletion Kit (which depletes the top 20 most abundant plasma and serum proteins).
PerkinElmer offers a slightly different twist to protein depletion in their new ProXPRESSION Biomarker Enrichment kits, which instead use cibacron blue to capture the carrier proteins themselves. This approach is based on the work of biomarker gurus Lance Liotta and Emanuel Petricoin of George Mason University, who demonstrated that low molecular weight protein fragments bound to these high-abundance proteins could represent biomarkers for disease.1 “We capture the high abundance proteins such as albumin, and then we elute the peptide and protein fragments that are bound to these carrier proteins and look at them in the mass spec,” says Lopez. The kits are compatible with PerkinElmer’s BioXPRESSION Biomarker Discovery Platform, a complete solution for top-down protein profiling centered on the company’s proTOF 2000 MALDI mass spectrometer.
For more general applications, Agilent’s new 3100 OFFGEL Fractionator helps reduce sample complexity by increasing the efficiency of protein fractionation and separation. The system is similar to 1D gel electrophoresis; however, fractionated proteins and peptides remain in solution above the gel rather than in the gel itself. Combined with reverse phase HPLC-Chip MS, “we find that the protein ID rate doubles over what we’ve done historically,” says Fjeldsted.
The final component necessary for high-throughput mass spec is software that can quickly analyze the large quantity of data coming off these new systems. PerkinElmer’s BioXPRESSION platform features software called Progenesis PG600 that can “compare thousands and thousands of high resolution mass spec profiles and very quickly find the significant differences, zero in on the masses, and give you the information, such as the P-values, how statistically significant they are, and how they cluster,” says Lopez. Likewise, Agilent’s new GeneSpring MS and MassHunter workstation were designed specifically for handling data from biomarker discovery studies.
References:
1. A.I. Mehta et al., “Biomarker amplification by serum protein binding,” Disease Markers, 19(1):1-10, 2003-2004.
