There are a number of flaws in this approach. One of the biggest flaws is that peak positions cannot be located accurately.
If the peak positions are not accurately and reproducibly determined, this inaccuracy can lead to substantial error both in
creating the calibration and in applying the calibration. If one examines a typical profile mode spectrum (Figure 2), it is
easy to see why there can be substantial errors in peak locations. First, there is substantial asymmetry in the peaks. This
asymmetry is related to the instrument "tune" and typically varies across the spectrum and from one instrument to another.
This makes determination of the peak position prone to substantial error, especially at low resolutions. Secondly, there is
the presence of noise, which can also lead to substantial errors.
Unfortunately, since the default mode is centroid-mode data acquisition, the data presented to the analyst have been processed
through various error-prone centroding algorithms. This results in significant and (most of the time) unrecoverable information
loss. It is therefore no surprise that the mass accuracy specified for conventional unit mass resolution MS systems is typically
no better than 0.1 or 0.2 Da.
A NEW APPROACH: CALIBRATING PEAK SHAPE
Based on these limitations, we concluded that calibration only on the X-axis would be insufficient to obtain the best mass
accuracy. Instead, a novel calibration approach was developed that not only calibrates the X-axis, but also calibrates the
peak shape. This is possible in MS because the theoretical peak positions are known very accurately (they are simply the mass,
or more precisely, m/z of each ion). In addition, we also know quite accurately the various isotopes and their relative abundances.
This information allows the calculation of a calibration function that corrects not only the peak position, but also the peak
shape.
We named this novel calibration technique "MSIntegrity," which calibrates the instrument to a symmetrical peak shape. This
method can dramatically improve all downstream processing. For example, we can perform accurate peak picking (the algorithmic
process of locating the center and integrating the area of a spectral peak) with no user-set parameters due to the calibrated
peak shape, which allows accurate peak center and peak area to be determined. As a side benefit, we found that noise can be
substantially suppressed without the loss of information and peak distortion typical of commonly used smoothing operations.
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Figure 3 illustrates the process of applying the MSIntegrity calibration. Although the process flow is identical to the conventional
approach, there are significant distinctions that preserve the rich information contained in a MS scan. A comprehensive calibration
involving both the mass and peak shape is ideally performed through on-board instrument processing to produce fully calibrated
MS data in profile mode. All downstream analysis, including accurate mass determination, will be based on this fully calibrated
MS data in profile mode. Alternatively, the calibration can be done after collecting profile mode data in the MassWorks software
implementation of MSIntegrity.
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