5 sample running problems highlighted by ion intensity maps

A typical LC-MS run, viewed as an ion intensity map (click to enlarge).

“What is an ion intensity map?” I hear you ask. At least, if you’re new to Progenesis QI, that may be your first question.

The ion intensity map (or “ion map” for short) that’s used in Progenesis is what many first-time users call a “2D gel-like representation” of their run. Indeed, it does look similar to a 2D gel, as seen in the image at the right.

In fact, what it shows is a map of ions in the run, with the darker areas showing the higher intensities in the MS signal. Retention time increases from top to bottom, while the mass/charge ratio increases from left to right.

The role of ion maps in quality control

A great benefit of this display is that it can offer an instant visual clue to a range of problems in your sample running. And by displaying them as soon as the data is loaded, Progenesis helps you to avoid wasting time downstream, trying to analyse data that should be rejected.

In this article, we’ll look at just a few of the sample running problems that the ion maps help you to spot.

1. The whole sample elutes over a short time-window

If all of your sample is eluting over a short time-frame within a long run, it’s likely that a higher proportion of your MS data will be without accompanying MS/MS data, when running in data-dependent acquisition (DDA) mode. The resulting lack of spectra will make it harder to identify the peptides.

2. Instability in the ion spray

Instability in the spray can be seen in the ion maps, manifested as gaps in the retention time axis of individual ions’ signals. This is clearer when you zoom into your runs. A consequence of the instability is that it can cause problems in peak picking and also result in increased quantification variance. You may need to check the spray for blockages or deposits.

3. A completely blocked ion spray

A more serious case of instability in the spray, this can be seen when there is a large gap in the retention times, meaning lots of peptides’ MS (and MS/MS) data is entirely absent. Again, you’ll probably need to re-run the sample after having cleaned the spray or replaced the LC column.

4. Contaminants and column-leaching

Dark, vertical stripes on an ion intensity mapLeaching of materials from the LC column is quite a common problem in LC-MS, although its severity varies greatly. Leaching tends to manifest itself in the ion maps in one of two forms: vertical streaks throughout your run; or diagonal streaking toward the end of your run. The latter is especially troublesome, as it is more likely to interfere with your data analysis results.

An ion map showing an extreme example of polymer leachingThere are many causes of leaching, so it’s impossible to prescribe any one solution for it. A common source of contamination, however, is from polymers like polyethylene glycol (PEG), which can leach from various plastics used in sample preparation. Other problems may arise from using inappropriate running conditions for your column, failing to clean a column fully between samples, or from precipitate building up in your column frits.

If you’re struggling, the Chromatography Forum is a good place to get advice from your peers. Waters also provide a very helpful troubleshooting guide (PDF).

5. Electronic noise in the MS signal

Electronic noise in the MS1 dataHopefully, this is a rare case. In this example, there’s a clear interference pattern in the MS data, visible throughout the entire run. The problem was eventually tracked down to a resonating, loose cable. If you see anything like this yourself, you’ll probably need to ask an engineer to take a look at your hardware.

Any more?

Have you seen any other unusual patterns in your MS data, when looking at the ion intensity maps? If you have, we’d love to hear from you and share your wisdom and experiences; just get in touch. And if you’ve not tried it yet, click here to download a trial of Progenesis QI for proteomics and start reviewing your ion intensity maps today.