Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Researchers often separate and identify molecules in a mixture using liquid chromatography-coupled mass spectrometry (LC-MS). Nano-LC-MS is a variant of the technique that uses a slower flow rate over a longer, narrower column to produce separations that are some 100 times more sensitive than conventional LC-MS.
The method is an important tool in proteomics, lipidomics, drug discovery and biomarker development, among other applications, and instrumentation manufacturers have developed multiple systems to support it.
Why nanoscale?
The main advantage of nano-LC-MS is sensitivity, says Evert-Jan Sneekes, strategic marketing manager for HPLC at Thermo Fisher Scientific. “Nano-LC provides the sensitivity that is required when the sample amount is very low, which is commonly the case in proteomics studies,” Sneekes says.
In part, that's because of the small size of nano-LC columns. Traditional LC-MS uses columns with internal diameters (IDs) greater than 1 mm. Nano-LC columns, however, hover around 75 μm. The smaller nano-LC columns enable users to apply smaller samples, which otherwise would need to be diluted below the detection limit. “The internal diameter of the [LC] column directly impacts the concentration of the sample, and smaller-internal-diameter columns yield less dilution and therefore enhanced sensitivity,” says Remco van Soest, technical marketing manager at Eksigent (part of AB SCIEX).
Nano-LC-MS also requires less material, which translates to a cost savings. Some simpler systems even allow users to pack their own nano-LC columns, which also saves money.
But there are disadvantages, too. Chromatography takes longer on nanoscale columns, and with its narrower columns and lower flow rates, the technique also is more prone to clogging.
Troubleshooting can be more complicated, as well. “Leaks are hard to detect, as the mobile phase will evaporate before being identified, so troubleshooting relies on monitoring system pressures,” says David Colquhoun, applications scientist at Shimadzu Scientific Instruments. “Consideration of cutting lines and making tight connections become more critical to prevent peak broadening and loss of signal.”
And nano-LC-MS tends to be less reproducible chromatographically, so research requiring longitudinal stability should use retention time standards, Colquhoun says.
New nano-LC-MS tools
Despite these concerns, new nano-LC-MS systems appear regularly in the market.
Agilent Technologies
Agilent’s 1260 Infinity Nanoflow LC system is suitable for proteomics and cellular research—for example, separating digests from gel bands, or peptides from complex protein mixtures. It is also appropriate for separating affinity-purified proteins that have post-translational modifications, for example in functional proteomics. Agilent’s Electronic Flow Control keeps the flow rate through the column stable by adjusting it in real time using active feedback. The system can be used for one- or two-dimensional LC and is compatible with most third-party and all Agilent mass spectrometers.
AB SCIEX
Available for both one- and two-dimensional (i.e., LCxLC) separations, Eksigent's NanoLC systems include the company’s patented Microfluidic Flow Control MFCPlus™ technology (which enables researchers to quickly and reproducibly change the solvent gradients within the column) and an autosampler for sample injections. Another offering, the cHiPLC® system, is an “easy plug-and-play chip-based platform” that can be used with the company’s ekspert™ nanoLC 400 system; the latter is currently used for high-throughput, high-performance proteomics analysis, including biomarker discovery, validation and verification, according to van Soest.
Shimadzu
Shimadzu’s Prominence nano LC system also offers two-dimensional LCxLC for greater resolving power. The two modes are independent, with all or part of the first (ion-exchange) column’s eluent being applied to the second (reverse-phase) column for further separation.
Shimadzu’s AccuSpot LC-MALDI Plate Spotter automates fraction collection from capillary or nano-LC columns. The AccuSpot continuously spots the LC eluent onto target plates that are used in MALDI-TOF-MS. “Nano-LC is most powerful when combined with mass spectrometry,” says Scott Kudzal, life science business manager at Shimadzu Scientific Instruments. He notes the combination can lend “a higher degree of precision and greater sequence coverage” to proteomics analyses.
Shimadzu also partnered with New Objectives to integrate that company’s PicoChip columns into Shimadzu’s LCMS-8030 and LCMS-8040 triple quadrupole mass spectrometers. PicoChip columns are all-in-one assemblies that contain both the nano-LC column and electrospray emitter (which delivers samples into the MS). This construction means less sample loss after the column and less peak broadening, because the sample is sprayed into the mass spectrometer right off the end of the column. This type of efficient sample loading is useful in proteomics and metabolomics analyses, in which sample volumes are limited and enhanced sensitivity is required, Kudzal says.
Thermo Fisher Scientific
Thermo Fisher Scientific offers two nano-LC systems for users with different experience levels or system demands. The EASY-nLC 1000 is designed for common nano-LC applications with minimal configuration. “Only the essential parameters must be set by the user, and the built-in intelligence allows an easy and efficient setup of the application,” says Sneekes. The company’s Dionex™ UltiMate™ 3000 RSLCnano system runs more advanced two-dimensional separations with complex samples. “The RSLCnano system provides more direct control over the run parameters, to optimize the conditions for the customer’s sample,” says Sneekes.
Thermo Fisher Scientific also offers a new type of coiled LC column with column lengths up to 50 cm. Like Shimadzu’s PicoChip columns, this design shortens the connection between the chromatography column and the mass spectrometer by integrating the column and the MS source into one unit. The Thermo Scientific EASY-Spray™ nano-LC-MS columns are also integrated with the MS source, but “the EASY-Spray columns do not have to compromise on the column length (and hence separation efficiency), because the column is coiled,” says Sneekes.
Waters
Waters’ ACQUITY UPLC M-Class nano-LC system performs two-dimensional LC using two reversed-phase columns, with the first-dimension separation running at pH 10 and the second at pH 2. The system also can be configured to perform hydrogen deuterium exchange mass spectrometry (HDX MS), which helps researchers study the dynamics of protein structure and conformational changes. The system’s extended pressure range (up to 15,000 psi) enables the use of column particles of less than 2-μm diameter, with 100 to 300-Å pore sizes. These features help the system separate sample constituents faster and with better peak resolution, allowing the detection of, for example, small changes in protein conformations.
Waters also introduced a line of 300-μm ID columns “that deliver true [nano-LC-MS] performance when configured with optimized flow cells for its ACQUITY optical detectors and a micro-probe for Waters electrospray sources,” says Patricia Young, senior marketing product manager at Waters.
The internal microfluidics of Waters’ ACQUITY UPLC M-Class System also were redesigned to minimize sample loss. “Many analytical scientists are faced with the task of extracting the most information from the smallest sample sizes,” says Young. “[They] need to reach lower and lower limits of detection and quantification on smaller and smaller sample sizes.”
Important nano-LC-MS considerations
When using nano-LC-MS, says Colquhoun, pay particular attention to sample preparation and column loading. Sample contaminants—such as detergents, lipids, salts or cellular debris—can clog nano columns more easily than their larger-diameter cousins. “Sample cleanup prior to introduction is critical,” he says. “A 2D system can alleviate many of these issues, as contaminants are diverted prior to introduction into the mass spectrometer.”
Other potential pitfalls also are magnified by the small internal diameters of nano columns. Dead volumes within the flow lines or poor connections between them—or even lines that are too long—can delay elution or broaden peaks, according to Colquhoun.
The separation powers of nano-LC-MS are impressive, but the technique comes with a catch: Learning to do it well requires significant time and effort. “A primary factor to consider prior to using nano-[LC-MS] is whether the increased sensitivity for sample characterization justifies the increased technical challenges and time investment involved,” says Jeremy Post, applications scientist at Shimadzu Scientific Instruments. “Learning and employing best practices for system plumbing and maintenance requires practice, patience and time.” But if your experiments demand extremely high sensitivity, it may well be worth it.
Image: The Thermo Scientific Dionex UltiMate 3000 RSLCnano System.