Hello!
I use Q Exactive mostly for intact mass analysis and peptide mapping of proteins in a range of 20-150 kDa and the software is Xcalibur.
During instrumental set up, I want to start using Lock Masses where I need to provide a mass (m/z) for internal calibration. There's a place where I can designate the lock mas (m/z) but I have a few questions regarding that:
1-Do people typically add a compound with known m/z to their sample for calibration using lock mass? Such as caffeine? If so what compound can be used for positive ions?
2-Can lock mass be one of the background masses? Or one of the m/z peaks that are related to buffers (m/z of products of formic acid, etc)?
3-Can this be used for both intact (MS) and peptide mapping (MS/MS) analysis?
4-When we enter a lock mass, will the software find the closest experimental m/z peak and matches and calibrates that to the theoretical m/z that we enter in the software?
I have a hard time finding experimental instructions for this online! I know these are simple questions but I'd appreciate your help!
Thanks!
Hello Daniel.
You can use a background mass, indeed. Some authors recommend choosing the [M+H]+ adduct of polydimethylcyclosiloxane C12H36O6Si6 (theoretical m/z 445.1200243) for positive mode, which could be suitable as it is a volatile contaminant found in many laboratories. I've just checked it, and polydimethylcyclosiloxane also shows up on my Q-Exactive.
However, you could really make use of any other known background mass, as long as it's not a casual interference but one that keeps appearing consistently. Luckily enough, Thermo provides a sheet containing possible background ions which could help you out.
I can't speak for others, but I infuse a calibration mix containing caffeine, Ultramark 1621, and some other compounds, for routine calibration in positive mode. I calibrate the Q-Exactive with this mixture in advance before analysing any of my samples, so no caffeine is added to my samples. In a nutshell, lock mass should be automatically performed in all your analysis using one of these background ions, but even if you use lock mass to correct m/z deviations, you should also calibrate your Q-Exactive periodically by infusing these calibration solutions. That's how you'll get an acceptable mass error without adding any calibrant to your samples.
Regarding question 4, if I'm not wrong that's what the software does unless you set a retention time window (Start and End cells in your screenshot). Nonetheless, background ions tend to appear throughout the mass spectra, so it won't be of much help.
Hope it helps!
In addition to Antonio Jesús Maldonado-Reina comments, for intact protein and peptide analysis, leucine enkephaline may be a good option that is typically used as infusing lock mass calibrant at TOF instruments.
https://www.waters.com/webassets/cms/library/docs/720001415en.pdf
- Badges
- Science topic