Shotgun lipidomics quantification

Quantification in shotgun lipidomics requires the addition of internal standards. Internal standards should be added during the extraction step and should consist of at least one standard for each lipid class. 

Yang, K., Cheng, H., Gross, R. W., and Han, X. 2009. Automated lipid identification and quantification by multidimensional mass spectrometry-based shotgun lipidomics. AnaZ. Chem. 81 4356-68. 

These principles of shotgun lipidomics can only be achieved in conjunction with the major feature of direct infusion, that is, ESI-MS analysis of lipids is conducted at a constant concentration of the solution. This feature in shotgun lipidomics provides many advantages for lipid analysis, particularly for the quantification of individual lipid species. Some of these advantages are as follows. First, constant interactions between lipid species are maintained under a constant concentration condition therefore, contribution of individual lipid species to the ion current in an ESI source is constant, thereby leading to a constant ratio of ion peak intensities between lipid species of a class. Such a constant ratio can be achieved under different experimental conditions (see Chapter 4), on different MS instruments, and in different laboratories. Second, also due to the constant interactions between lipid species under the condition, ion suppression between each other within a lipid class or between lipid classes is constant. Third, lipid aggregation, which is a big concern for lipid quantification, can be well controlled and minimized. 

The third factor is to set up the MS (or MS/MS) parameters to identify and quantify the eluted individual lipid species as many as possible. The particular features in LC-MS analysis are that the lipid concentrations in eluents are constantly changing, and identification and quantification of lipid species have to be done in a very limited time frame. These features are in contrast to shotgun lipidomics therefore, totally different settings and methodologies from those in shotgun lipidomics have to be employed. 

Wang, M., Hayakawa, J., Yang, K. and Han, X. (2014) Characterization and quantification of diacylgjycerol species in biological extracts after one-step derivatization A shotgun lipidomics approach. Anal. Chem. 86, 2146-2155. 

S LipidSearch LipidSearch is the commercial software (Thermo Fisher Scientific) developed jointly by Prof. Ryo Taguchi and MKI (Tokyo, Japan). It is a powerful new tool for automatic identification and relative quantification of cellular lipid species from a large amount of mass spectrometric data obtained from both LC-MS and shotgun lipidomics approaches. A lipid database containing... 

For those who would like to employ shotgun lipidomics (particularly MDMS-SL) for identification and quantification of individual species of a class, familiar with the fragmentation patterns of individual lipid classes, could enable them to design MS/MS scans for building-block analysis in the PIS, NLS, or both modes to selectively identify individual molecular species of a lipid class of interest. 

Although the requirement of the internal standard levels for LC-MS analysis of lipids is not as strict as shotgun lipidomics, recognizing the effects of internal standard levels on accurate quantification is still useful and should be integrated into the method development or validation. For example, the level of an internal standard in LC-MS analysis also should not be too low since a lower level of an internal standard makes the experimental error inherited with the internal standard to be amplified as in shotgun lipidomics. 

Many lipid classes can be quantified by this improved shotgun lipidomics approach. Quantification of individual species of a lipid class is conducted based on the summed abundance of its major fragment(s) in comparison to the counterpart of the spiked internal standard of the class. The effects of different isotopologue... 

Note that this shotgun lipidomics approach is based on tandem MS analysis. As discussed earlier, due to the differential kinetics and/or thermodynamics of different species of a class during CID, two or more internal standards of a class spiked during lipid extraction are necessary to minimize any effects of differential fragmentation patterns on quantification. This point is particularly important to those species containing polyunsaturated fatty acyl substituents [20]. Moreover, identification and quantification of low-abundant isomeric/isobaric species overlapped with an abundant species may be missed due to the limitation of a relatively narrow dynamic range. 

It is worthy to note that when only two species, including the preselected internal standard, meet the criteria for selection as the standards for the second step of quantification in MDMS-SL, this second step of quantification is virtually identical to that used in tandem MS-based shotgun lipidomics with a linear standard curve [24]. In this case, the presence of different numbers of double bonds might affect the accurate quantification of those overlapping and/or low-abundant species performed in the second step of quantification. This effect is relatively small in MDMS-SL, especially for the total content of the class, with the following reasons (1) the selected, endogenously obtained standard usually contains a certain number of double bonds and (2) the species determined in the full MS mode in the first step of quantification largely contributes to the total content of the lipid class of interest. 

There is another big difference between the second step of quantification in MDMS-SL and that used in tandem MS-based shotgun lipidomics. All quantified... 

Finally, MDMS-SL possesses other advantages in comparison to the tandem MS-based shotgun lipidomics. For example, the second step of quantification in MDMS-SL can apply any or all head group-related PIS and/or NLS of the lipid class, if present and sensitive enough, for quantification of individual species in the second step. This redundant process is very useful to refine the data and serves as an internal validation of the determined results. Moreover, with the second step of quantification, an over 5000-fold linear dynamic range for many lipid classes can be readily achieved [36] due to the second step of quantification serving as a relay for the dynamic range. 

First, it is always better to optimize the collision energy that can balance the fragment intensities of all the ions of the entire class of interest for quantification of lipid species by tandem MS in shotgun lipidomics even more than one internal standards are employed in the method. Similarly, optimization of the SRM/MRM conditions for individual species in an LC-MS/MS method is not recommended when interest is to quantify all the species of a class, unless a calibration curve for each individual species is established under the identical conditions, since optimization of MRM conditions for individual lipid species leads to an incomparable response factor of the species of interest to that of the selected internal standard. In both cases of shotgun lipidomics and LC-MS/MS analyses, different collision energies applied for different species could lead to substantial errors in quantitative analysis, as discussed previously [22]. Careful attention to CID energy must be exercised if accurate quantification is a goal. 

Actually, a similar phenomenon to this steady-state ion suppression in shotgun lipidomics is also present in any method developed with LC-MS for quantitative analysis of lipid mixtures. For example, if it is intended to quantify a species of a minor lipid class in the presence of other abundant species [24], the amount of total lipids that can be loaded onto a column are capped by the upper limit of the linear dynamic range of the most abundant species in the mixture under the experimental conditions. The loaded amount of total lipids to expand the linear dynamic range of the minor component in the method cannot be increased greatly if there is a need for quantification of major components as well. Of course, the minor species can be analyzed separately with a pre-isolated fraction or with a saturated concentration of the abundant species to increase the dynamic range for quantification of the minor components. 

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