Send to printer »

Tutorials : Sep 15, 2008 (Vol. 28, No. 16)

Improving High-Res Mass Spec Formula ID

Line-Shape Self Calibration Can Be Used to Fine-Tune Mass Accuracy Measurements
  • Don Kuehl

Mass spectrometry (MS) has become a fundamental tool for compound identification or confirmation by virtue of its ability to perform elemental composition determination (formula ID) by accurate mass measurements. The speed, sensitivity, and ease of interfacing the technique with gas and liquid chromatographs make MS the technique of choice for many applications.

In addition to accurate mass measurements, the isotope abundance distribution for an ion also provides information unique to a given chemical formula. The mass spectral accuracy required for accurate isotope modeling, however, has previously not been easily obtainable.

Mass accuracy is the measure of an instruments error in determining the theoretical (exact) mass of an ion. It is usually expressed in parts per million relative to the measured mass values or in absolute units of milli-Daltons (mDa) (Figure 1). Every unique formula has a unique mass value, which is the basis for formula ID using accurate mass measurements.

Unfortunately, due to measurement error, mass accuracy alone can rarely provide a unique formula, particularly at higher mass values (above 400 Da). For example, the formula search on an instrument capable of obtaining a mass accuracy of 1 ppm results in a list of 34 formula candidates for an unknown compound at 500 Da containing the elements C, H, N, O, S, and Cl.

The formula candidates can be pared down by imposing chemical constraints such as: limiting the possible elements in the formula search, restricting the minimum and maximum number of atoms for each element, electron state, and utilizing any other complementary knowledge of the unknown sample. For true unknowns, even on the highest resolution instruments such as FT-ICR systems, mass accuracy alone is not enough to identify a unique formula.

An ion’s isotope pattern is also unique for each formula and significantly richer in information than the measurement of a single peak position, as with accurate mass measurements. It is composed of many peaks, each with unique relative intensities based on their isotopic abundances and unique relative mass positions based upon the mass of each isotope.

Spectral Accuracy

Spectral accuracy is a measure of the similarity between the measured spectra (the entire ion’s isotope pattern) against that of the theoretical spectrum. Since the line-shape of the measured spectrum is unknown, measures of spectral accuracy are generally not high enough to provide a unique formula ID (Figure 2).

This limitation can be elegantly addressed by simply calibrating the line-shape to a mathematically defined function. This allows extremely accurate comparisons to be made between measured and theoretical spectra with spectral accuracy values capable of uniquely identifying an unknown ion formula (Figure 3).

To perform line-shape calibration on high-resolution instruments, one must first measure a single pure line from which the proper correction function can then be calculated. For this, the fully resolved monoisotopic peak (Figure 2) from the high-resolution measurement, which by definition is a single pure isotopic line, is used.

Once the calibration function is calculated, it can then be applied across all of the isotope peaks of the analyte ion (M+1, M+2, etc.).

Because the calibration function is being calculated without a known standard, it does not have any effect on improving the mass accuracy, and it is solely a correction of the instrument line-shape.

But once the ion is calibrated to a known line shape, highly quantitative and accurate comparisons between isotope distributions can be used to improve the discrimination of formula candidates derived by mass accuracy alone.

The comparison results in the interpretable statistic of the root mean square error (RMSE), which can be converted to percent spectral accuracy as:

(1-RMSE)/S*100

where S is the level of measured ion signal.

This line-shape calibration approach has been named sCLIPS, or self calibrating line-shape isotope profile search, by virtue of the fact that the unknown ion itself is used to create the line-shape correction.

The sCLIPS approach is easy to apply to any high resolution instrument including time-of-flight, OrbiTrap, FT-ICR, high-resolution quadrupoles, as well as magnetic sector instruments. It can provide substantial improvements for formula ID, and in many cases can eliminate a large number of formula candidates derived from an accurate mass-only search. sCLIPS is a feature of Cerno Bioscience’s MassWorks software for mass spectrometery.

Additional benefits of MassWorks sCLIPS include the ability to help identify situations where the formula candidates are likely to be incorrect or where other fundamental measurement problems such as interferences or detector saturation exist. These situations are readily identifiable since they distort the spectral isotope patterns that directly impact the quality of the spectral accuracy match, something that is not possible to do when using mass accuracy alone.

Line-shape self calibration is particularly applicable to the analysis of unknown small molecules in areas such as natural product analysis, drug or product contaminants, impurities, or degradents.