Intrasource separation

Figure 12.18. Shotgun lipidomics using ESI intrasource separation and multidimensional mass spectrometry of lipids from a complex extract. (From refs. 65 and 66.)...

Through global lipidomics, each distinct lipid species present in a cell s lip-idome can be identified. A shotgun lipidomics approach, which uses an ESI-intrasource separation of Upids from a complex extract, multidimensional mass spectrometry, and computer-assisted array analysis, is described. 

What is the basis of an ESl-intrasource separation of lipids into distinct... 

Outline the ESI intrasource separation-multidimensional MS lipidomics approach for profiling lipids in complex biological extracts. 

Such a separation of different lipid classes in the ion source (which is termed as intrasource separation [20]) is similar to the use of an ion-exchange column [21] or an electrophoretic device to separate individual lipid classes. However, compared to ion-exchange chromatography and electrophoresis, the intrasource separation has many advantages such as rapid, direct, in situ, reproducible, avoiding artifacts inherent in chromatography-based separations [22]. 

Features of Electrospray Ionization for lipid Analysis In addition to intrasource separation for lipid analysis, there exist numerous other features of ESI for lipid analysis as follows ... 

The differential charge properties of different lipid classes (which are predominant with the head groups of polar lipid classes) are exploited to selectively ionize a certain category of lipid classes under multiplexed experimental conditions to separate many lipid classes in the ion source (i.e intrasource separation) [33]. This separation method is analogous to the electrophoretic separation of different compounds that possess different pi values [33] (see Chapter 2 for details). 

Figure 4.8 Schematic comparison of intrasource separation of lipid categories to the theoretical electrophoretic separation of lipid classes, (a) Schematically shows the selective ionization of different lipid categories under three different experimental conditions with or without adding a small amount of LiOH. (b) Schematically shows the imaginary chromatograms of lipid classes after electrophoretic analyses under corresponding experimental conditions. PC, TAG, FA, PE, and AL stand for phosphatidylcholine, triacylglyceride, nonesterified fatty acid, phosphatidylethanolamine, and anionic lipids, respectively. Christie and Han [lb]. Reproduced with permission of Elsevier.

Figure 4.9 Representative ESI-MS analysis of lipid classes resolved by intrasource separation. Lipid extracts from mouse liver samples were prepared by using a modified procedure of Bligh and Dyer [1]. MS analysis was performed with a TSQ Vantage triple-quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an automated nanospray apparatus (i.e., TriVersa, Advion Bioscience Ltd., Ithaca, NY) and Xcalibur system software. Mass spectra were acqnired directly from the diluted hpid extract in the negative-ion mode (a), after addition of 50 nmol LiOH/mg of protein in the diluted lipid extract and analyzed in the negative-ion mode (h), or the identical hpid solution to that in (b) in the positive-ion mode (c). IS denotes internal standard PC, PE, PG, PI, PS, TAG, NEFA, and CL stand for phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidyUnositoL phosphatidylserine, triacylglycerol, nonesterified fatty acid, and doubly charged cardioUpin, respectively.

Collectively, IM-MS clearly provides separation of lipid classes according to their charge properties, individual molecular species of a lipid class based on their molecular size (including chain length and unsaturation), and isobaric/isomeric species possessing different conformational structures [83]. This in situ drift time/collision cross section variation could be used as an additional variable to the other separation variables (e.g., intrasource separation, LC-MS elution, and optimal selection of MALDI matrix for ionization) described earlier as an aid to providing 3D analysis of complex lipid mixtures. 

A low-concentration of lithium chloride (e.g., 50 mM in aqueous phase) is preferable for lipid extraction for MDMS-SL since the lithium adduct of lipid species can yield unique and informative fragmentation patterns after CID (Part II). Moreover, the weakly acidic conditions resulted from a weak Lewis base of lithium ion and a strong Lewis acid of chloride could improve the extraction efficiency of anionic lipids to a certain degree and could lead to increased intrasource separation of molecular specie of anionic lipid classes from PE species in negative-ion ESI-MS (Chapter 3). Under such weakly acidic conditions, degradation of plasmalogen species does not occur. 

After separation of different lipid classes in the ESI source (i.e., intrasource separation) and MDMS identification of individual species (see Section 3.2.3.3), quantification of the identified individual species of a lipid class of interest is performed in a two-step procedure in MDMS-SL [11, 35]. This procedure can be conducted automatically [29]. 

Han, X., Yang, K. and Gross, R.W. (2008) Microfluidics-based electrospray ionization enhances intrasource separation of lipid classes and extends identification of individual molecular species through multi-dimensional mass spectrometry Development of an automated high throughput platform for shotgun lipidomics. Rapid Commun. Mass Spectrom. 22, 2115-2124. 

Multidimensional MS-Based Shotgun Lipidomics after Intrasource Separation... 

Figure 20.6. Intrasource separation of a mixture of phospholipids. The phosphohpid mixture is comprised of 15 0 15 0 and 22 6-22 6 GPGro (1 pmol/pL each), 14 1-14 1 and 18 1 18 1 GPCho (lOpmol/pL each), and 15 0 15 0 and 20 4—20 4 GPEtn (ISpmol/pL each) in 1 1 CHCls/MeOH. Panels A and C show mass spectra acquired in the negative-ion mode and Panels B and D show mass spectra acrjuired in the positive-ion mode in the absence (Panels A and B) or presence (Panels C and D) of LiOH. The hmizontal bars indicate the ion peak intensities after C de-isotoping and normalization of molecular species in each class to the one with lower molecular weight.

After separation of different lipid classes in the ESI ion source (i.e., intrasource separation) and identification of individual molecular species by multidimensional MS, quantitation of... 

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