Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Mass spectrometry (MS) enables researchers to investigate a host of properties, such as a sample’s molecular weight, structure, identity, quantity and purity. When coupled with a sample preparation technique like liquid chromatography (LC), LC/MS becomes a powerful analysis tool capable of extracting meaningful data about a range of compounds, including drug metabolites, peptides and proteins from complex biological mixtures like milk, serum and whole-cell lysates.
Just as LC complements MS by preparing the sample, MS in its simplest form acts as the LC detector, providing a readout of the chromatographic separation. In some cases, LC can separate compounds with the same mass/charge ratio (m/z) that MS is unable to separate; in others, MS can distinguish compounds that co-elute from chromatography columns.
LC/MS
Before samples are introduced into an MS instrument, they’re typically prepared by filtering out particulates, concentrating the analyte, desalting and generally separating out compounds that may cause background ions or suppress ionization. [1] Most often, many or all these steps are accomplished by HPLC, UPLC or even miniaturized (nano-)systems that can operate at high pressures/low flow rates and that allow users to streamline the workflow by directly interfacing, online, with the MS via an ionization device.
Early incarnations of ionization devices required samples to be ionized under vacuum, severely limiting the selection of compounds that could be analyzed. Newer techniques, such as electrospray ionization (ESI), now enable ionization to take place at atmospheric pressure, greatly expanding the repertoire. Once ionized, analytes—ions resulting mostly from the addition or loss of water or a proton or an electron —are mechanically and electrostatically separated from the neutral (uncharged) molecules. Depending on the configuration of the instrument, either all or selected subsets are allowed through to a detector that charts the m/z of the ions relative to their signal strength (which indicates abundance).
Tandem MS
The m/z information obtained from single-stage MS is very valuable and sometimes can be used to identify small molecules. But for larger, more complex molecules, complementary structural information generally is needed.
Analyzing a complex mixture of proteins by LC/MS typically starts with an overnight trypsin digestion to create peptides, “and then the proteins are identified at the peptide level by collecting tandem MS data,” says Larry David, Ph.D., director of the proteomics shared resource at Oregon Health and Science University (OHSU). “Because masses alone are usually not good enough to identify a peptide.”
In tandem MS, a sample sequentially traverses different stages of the instrument, each subjecting it to a process such as selection or fragmentation. In a triple-quadrupole MS, for example, the first stage selects a particular m/z range, discarding the rest. These remaining ions are then fragmented by colliding them with neutral molecules in the second stage. The newly fragmented ions then go into a third stage, which either scans the spectrum of product ions or selectively monitors just a few, depending on the experiment. These spectra are then compared to a database of actual or theoretical spectra to determine their identity, enabling researchers to piece together the parent molecules they came from.
By repeating the tandem MS process over and over—that is, iteratively fragmenting the ions, generating a spectrum and selecting the new ions to be re-fragmented—complex molecules like proteoglycans can be serially deconstructed and computationally reconstructed until their molecular structures have been determined.
Other uses of LC/MS
The power of LC/MS frequently is brought to bear on identifying protein modifications such as phosphorylation and di-sulfide bridging, as well.
Between the separation afforded by LC and the ability of tandem MS to discard nonanalyte ions, LC/MS is the choice for many different applications. For example, David frequently does experiments to determine what proteins are present in complexes, using immunoprecipitation to find the protein-protein interactions. “It’s much easier” to analyze with LC/MS than with other techniques, he explains.
David’s proteomics core also uses the extraordinary sensitivity of LC/MS to undertake some less common structural analysis techniques, such as hydrogen-deuterium exchange, probing the sites where proteins are exposed to solvent.
David thinks that using LC/MS strictly for proteomics is “probably just the tip of the iceberg.” He points out that OHSU has a second core that does LC/MS only on small molecules—for example, to query small-molecule metabolites like ATP, phospholipids, or intermediates in purine metabolism. “And then we do all the protein analysis in our core.”
Reference
[1] Basics of LC/MS Primer, Agilent Technologies, 2001.
The image at the top of the page is from Agilent Technologies.