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Mass Spectrometry and Liquid Chromatography: A Dynamic Duo


by Josh P. Roberts


ass 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 meaning- ful data about a range of compounds, including drug metabolites, peptides, and proteins from complex biological mixtures such as milk, serum, and whole-cell lysates.


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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 coelute from chromatography columns.


LC/MS Before samples are introduced into an MS instrument, they are typi- cally 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 of


these steps are accomplished by HPLC, ultrahigh-performance liquid chromatography (UHPLC), or even miniaturized (nano-) systems that can operate at high pressures/low flow rates and that allow users to streamline the work flow 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 me- chanically 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, Portland). “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 fragmen- tation. 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 iden- tity, 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 refragmented—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 disulfide 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 applica- tions. For example, David frequently does experiments to determine


AMERICAN LABORATORY • 34 • JUNE/JULY 2013


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