What Kind of Mass Spec User are You?

1. The Down and Dirty Protein Chemist

The protein chemist just wants to know what it is that he's looking at. He could be looking to understand the biology of a protein by characterizing its coimmunoprecipitation partners, or identifying a particular spot on a two-dimensional gel. For these kinds of applications a quick and dirty approach will generally give the answers he needs.

Recommended System: MALDI+TOF

Reason: Peptide mass fingerprinting and MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry is a reasonable first approach, according to Neil Kelleher, associate professor of chemistry at the University of Illinois, Urbana-Champaign.

The advantages of TOF, says Patrick Limbach, professor of chemistry at the University of Cincinnati, are that it is a simple mass analyzer configuration with good sensitivity and "a broad mass analysis window" (meaning that TOF can analyze fragments from 10 to hundreds or thousands of atomic mass units). Another advantage, he says, is the analyzer's speed. "That's why it works well with MALDI, because you can run at a high repetition rate on the laser and acquire lots of spectra per second with the TOF."

MALDI is a reasonable first-pass approach, says Michael Gross, professor of chemistry at Washington University and editor of the Journal of the American Society for Mass Spectrometry, but it isn't for everyone. "If in your coimmunoprecipitation you have 20 or 30 proteins, each giving 50 tryptic pieces, you have 1,000 pieces." MALDI, he says, "will never pop all of them in the gas phase." To get more information, consider a tandem configuration that can provide sequence detail, such as MALDI-TOF-TOF, or even a more sensitive device, such as an ion trap.

2. The Sensitive Type

The devil is always in the nitty-gritty details, and for proteins, that means posttranslational modifications. Suppose, for instance, that you're looking at histones, which can bear both acetyl and trimethyl modifications. Both moieties produce nominal mass increases of 42, and a standard mass spec cannot distinguish the two. A high-mass-accuracy instrument can, however, since it can report masses to between two and four decimal places.

Recommended System: LC+ESI+FTICR with ECD

Reasons: High-mass-accuracy instruments can be used to differentiate molecules that appear identical with so-called "nominal-mass" instruments, says Gross. Your best bet to find out would be to use liquid chromatography (LC) coupled to an electrospray ionization (ESI) source and an FTICR mass spec featuring high-mass-accuracy and sensitivity. You'll also want to use electron capture dissociation (ECD) for your tandem work.

Though the typical collision-induced dissociation (CID)-mediated approach to tandem mass spec can spot modifications, it isn't really ideal for identifying which residue actually bears the modification, according to Limbach; the dissociation event often shaves the modification from the peptide as it fragments the peptide itself. ECD, however, leaves modified residues intact. The technique only works on FTICR instruments at the moment, though work is underway to bring it to lower cost instruments as well, says Limbach. But there's a caveat: these instruments have such tight tolerances they can often miss the unexpected, such as deamidation of asparagine residues, or phosphorylation events.

3. The Outsider

Not everyone is interested in proteins. You might want to know, for instance, if a particular nucleic acid contains unusual or modified residues (such as methyl-C), and if so, where in the sequence they are located. Both questions may be addressed using an LC-ESI-tandem mass spec (such as a QTOF or Qtrap configuration); the former in negative-ion mode (because of the nucleic acid's negatively charged backbone), and the latter in positive mode.

Reason: Limbach does his nucleic acid work using both an LC-ESI-linear ion trap and an LC-ESI-QTOf (quadrupole-time of flight hybrid). "It's not mission-critical which particular tandem mass spectrometer you have, but you want to have the tandem MS capability," he says - both to identify modifications, and also to determine where in the polynucleotide chain the modification is located. In tandem-mass spec mode, ions are measured, degraded (e.g., with CID or ECD), and then measured again to provide sequence or structural information. Yet tandem instruments are not all the same. John Yates, professor of cell biology at the Scripps Research Institute in La Jolla, Calif., says that linear ion traps are fast, "much faster than a QTOf, but the resolving power and mass accuracy is less." Both are relatively inexpensive options, however, when compared to FTICR instruments. These offer the best mass accuracy around, but also are relatively slow and insensitive, require a superconducting magnet, and demand "sophisticated" operators.

4. The Mixer

Sifting through small-molecule metabolites (sugars and lipids, for example) requires a different set of instrumentation considerations. You might be operating in discovery mode, looking for a biomarker for a particular disease, say, or drug efficacy. In that case, you'll need tandem mass spec capabilities to nail down chemical structure, and your instrument of choice is an LC-ESI-triple quad. More importantly, however, you'll need multiple ionization methods to cast the widest net. Consider using the two electrospray variants, APPI (atmospheric pressure photoionization) and APCI (atmospheric pressure chemical ionization).

Recommended System: LC+ESI+triple quad with multiple ionization sources

Reason: APCI uses the chemical components in the solvent to ionize a sample, says Limbach. "Let's say I have a sample in a methanol/water solution. [APCI] will use either the methanol or water in a chemical reaction to ionize the sample. In APPI, you shine light on the sample to photochemically generate an ionization." And just as MALDI and electrospray cannot ionize precisely the same molecules, neither can APCI nor APPI.

5. The Counter

Once you've identified your biomarker, you now need to count it, perhaps in hundreds or thousands of biological samples. The go-to mass analyzer for quantitative applications is the triple quad, which you'll want to couple to liquid chromatography and an electrospray ionization source. "The triple quads, their bread and butter is really quantitation," says Gafken. "There are many ways to quantitate proteins and peptides, but if you need absolute quantitation, the best method is a triple-quad instrument."

Reason: "Quadrupoles are really filters; they operate like a radio," Limbach explains. "You tune a frequency, and only a particular ion comes through, and you scan the radio dial to get the ions out. Everything else that isn't the right mass hits the wall and is lost." These instruments excel in scanning one or perhaps two ion frequencies (that is, mass-to-charge ratios, or m/z), but are too slow for the fast m/z scanning that is required in most discovery mode work.

But how do you know the ions you see are the ions you want? In any biologic sample, several ions might have the same m/z value. That's where single-reaction monitoring comes in. "That overcomes the slow speed of that first quad and the scanning, because you know what you're looking for," says Gross. So if, for instance, your particular molecule has m/z 1000 and fragments into an ion of m/z 300, you can set the first quad to filter ions of m/z 1000, fragment them in the second quad, and count the fragment ions of m/z 300 in the third quad. In multiple reaction monitoring, you set the instrument to "hop" from one m/z value to another, thereby enabling you to count two, or perhaps three analytes simultaneously.