Ask the Editor: Maximizing the Use of a Single-Quadrupole MS System

The answer to the following question was provided by LCGC’s “MS — The Practical Art” columnist Michael Balogh.

Q: This question is about the versatility of a single-quadrupole system for high molecular weight work. In other words, how can I maximize the use of a single-quadrupole mass spectrometer?

A: If a single-quadrupole system — or a tandem system for that matter — demonstrates good “electronic” (and thermal) stability, the mass assigned in calibration will remain dependable. Relaxing the typical resolution for quadrupoles to resolve isotopes (0.6 Da full width at half-height of a given peak) is a good technique considering that high molecular weight analytes may have little or no interference surrounding them. An early article on resolution and mass accuracy from “MS — The Practical Art” column in LCGC might be useful (1).

Since we are often working in full profile acquisition mode in high mass acquisitions (not reducing the spectra to stick plots) smoothing the peak top may prove useful. Cerno Bioscience (Danbury, Connecticut) has developed spectral shaping software that requires a profile or continuum trace input, which may improve results here as well especially at low signal-to-noise ratios.

More specifically here are some comments I adapted some time ago from Brian Green, long-time researcher and developer with Micromass (Manchester, UK) who is a leading practitioner in the art of high mass accuracy on quads:

Low-resolution quadrupole systems are also used in extremely high mass accuracy measurements with analytes such as proteins. The masses of proteins are generally defined as “average” values when the isotope peaks are not resolved from one another. Average mass is the weighted mean of all the isotopic species in the molecule. The instrumental resolution normally employed on quadrupole instruments a 10-kDa protein broadens by a factor of 1.27. This increases significantly as the mass increases (that is, to a factor of 2.65 at 100 kDa). However, by reducing the peak width to mass-to-charge ratio (m/z) 0.25 (increasing resolution to 4000), the situation is dramatically improved. Many quadrupoles can achieve this performance, as can all the time-of-flight instruments with ease.

In practice, electrospray ionization mass spectrometry (ESI-MS) produces multiply charged ions. Hence, the widths need to be divided by the number of charges on an ion in order to give the width on the m/z scale. For example, a 20-kDa protein with 10 or 20 charges on it will produce isotope envelopes that are 0.9 or 0.45 m/z units wide at m/z ~2000 or ~1000 respectively.

When these ions are observed on an instrument set for a significantly lower resolution than is required to resolve the isotopes (say less than 10,000 resolution), a single peak is produced for each charge state. The overall width is determined by combining the instrumental peak width with the theoretical width of the isotopic envelope divided by the number of charges on the ion. The instrumental peak width would be determined on the first isotope peak of a low molecular weight compound at the same m/z value as the multiply charged protein peak.

Reference: (1) M.P. Balogh, LCGC 22(2), 118 (2004).

Questions?

LCGC technical editor Steve Brown will answer your technical questions. Each month, one question will be selected to appear in this space, so we welcome your submissions. Please send all questions to the attention of "Ask the Editor" at lcgcedit@lcgcmag.com We look forward to hearing from you.