Huan He, Ryan P. Rodgers, Alan G. Marshall, and Chang Samuel Hsu

OUTLINE REVIEW

PART 2 OF 2

OUTLINE OF RESULTS AND DISCUSSION

HPLC to Address Concentration Dynamic Range

• LC separation of the polar lipid fraction is essential for lipid characterization by MS; direct infusion of the polar lipid fraction of algae yields only nonlipid signals.  LC separation also increases the number of detected lipids by MS.
• Only 2.4 ug of dry algae for each nLC-MS experiement was required for nano-LC
• Figure 1 shows the LC separation of several representative polar lipids, and only a relative few peaks were selected for identification with further mass spectrometry:
  • The monoacyl lipids (LPE, LPC, LPI, SQMG, FA, ...)
  • C₃₂H₆₆O₈S₂
  • IPC (inositolphosphorylceramide)
  • C₃₂H₆₄O₅S₁
  • SQDG (sulfoquinovosyldiacylglycerol) (16:1/16:0)
  • PI (phosphatidylinositol) (16:1/16:0)
  • PG (phosphatidylglycerol)
  • DGDG (digalactosyldiacylglycerol) (20:5/16:1), (20:5/16:0)
  • C₃₂H₆₂O₄S₁
  • MGDG (monogalactosyldiacylglycerol)

• The nLC-MS experiment detected nearly 200 unique lipid species (not counting various adducts) with a dynamic range greater than 20,000:1.

Identifying components from Nannochloropsis occulata with MS:

• Two main types of MS exist: destructive and non-destructive.  In destructive MS, the sample is bombarded with an electron beam while the ion charge to the sample is constant (usually at a charge of -1), and the molecules breaks apart into several fragments.  Non-destructive MS varies the charge of the molecule, but the molecule is not dissociated.  FT-ICR MS offers a partial non-destructive method of identifying components of a sample, as so done with Nannochloropsis oculata by a cyclotron particle accelerator.  In a cyclotron, the particles are held to a spiral trajectory by a static magnetic field and accelerated by rapidly varying the electric field.  The actual motion of the particle by the FT-ICR MS is a helix.  The molecule is dissociated at oxygen linkages.
  http://en.wikipedia.org/wiki/Ion_cyclotron_resonance
• Figure 2 shows a close-up section of a MS spectrum, showing the precursor ion SQDG.
  • The resolution (R, m/Δm_50%), necessary to resolve between ³⁴S and ¹³C₂, can be calculated from the figure with the resolving power Δm_50% (obtained from the inset of the figure, 11.0 mDa):
    
    • 793.5 Da/(793.5053 Da - 793.4943 Da) = 793.5 Da/0.011 = 72136 = R required for observing two distinct peaks
    • The actual FT-ICR MS data was acquired at a mass resolution of 200,000 at 400 Da.  Actual resolving power (Δm) calculates to be 0.002 Da.
  • Detection of the two distinct peaks at nominal m = 793.50 is possible by the formula m/z = eB/2πf, where f is the cross-sectional frequency of a helix in the case of FT-ICR with LTQ, being the path of the molecule.  The SQDG molecules of ³²S,
    ¹³C₂ are isotopically heavier than the SQDG of ³⁴S, ¹²C₂.  The heavier particles will have a slower motion than the lighter particles, so the former will have a lower frequency than the later.  The difference in frequency causes separate peaks to appear in the data.
  • The separation of the two peaks at the nominal 793.5 can be calculated by the masses of ³⁴S, ¹²C₂ and ³²S, ¹³C₂.  The unit Dalton (Da, or u) was used to show the mass difference.  The calculation for finding the peak separation, and confirming the presence of sulfur, is as follows:
    
    • ³⁴S = 33.967861176 u
    • ¹²C = 12.000000000 u
    • ³²S = 31.972070694 u
    • ¹³C = 13.003350258 u
    By definition Carbon-12 is 12.000000000 u, and the values of ³⁴S, ³²S, ¹³C₂ accounts for the problem of mass defect.  Note that the mass of an atomic nucleus is always less than the sum of the individual masses of protons and neutrons.  The mass defect is due to the nuclear binding energy of the nucleus, which can be described by Einstein's famous equation E = mc².
    
  • Calculate the difference between ³⁴S, ¹²C₂ and ³²S, ¹³C₂:
    
    • ³²S + ¹³C₂ - (³⁴S + ¹²C₂)
    • = [31.972070694 + 2*(13.003350258)] - [33.967861176 + 2*(12.000000000)]
    • = 0.01091002 Da = 10.91002 mDa
  
• Figure 3 shows the overall MS of deprotonated sulfoquinovosyldiacylglycerol (SQDG) - collision-induced dissociation (CID) spectrum, with a minor peak at 791.4985 described by Figure 2.  The other peaks represent the fragment masses of SQDG, with the dissociation pattern described by showing a molecule in the inset of the figure using arrows to identify bond breaking, and labeled with the fragment mass.

• Figure 4 displays the lipids identified from Nannochloropsis oculata.  The structure of the individual lipid species is determined by accurate mass of each pseudomolecular ion by FT-ICR MS and LTQ CID mass spectrum.
• Figure 5 displays the relative mass spectral peak magnitudes for the identified lipids:
• Figure 6 identifies the molecular ions C₃₂H₆₂O₄S, C₃₂H₆₄O₅S, and C₃₂H₆₆O₈S₂ with confirmation of the presence of sulfur.
• Figure 7 shows the polarity order corresponding to the retention time order for C₃₂H₆₂O₄S, C₃₂H₆₄O₅S, and

  C₃₂H₆₆O₈S₂

• Figure 8 is the first ion mass spectrum in this article where the charge, z, is greater than 1-, characterizing the bisulfur compound C₃₂H₆₆O₈S₂ by the fragment ion [HSO₄]^-
• Figure 9 displays signal magnitudes of SQDG, IPC, MGDG, and DGDG variants, distinguished by different hydrophobic tails (R_x).  All the lipid isoforms are presented at A : B.  A is the total carbons of the diacyl or monoacyl chains; B is the total number of double bonds in the diacyl or monoacyl chains.  For lipids with diacylglycerol backbone, A : B only gives the sum of the total carbons and double bonds of the two acyl chains.  A : B does not specifically identify individual acyl chains, and it can be a mixture of isomers.  For example, SQDG (32 : 1) can be a mixture of SQDG (16 : 1/16 : 0), (16 : 0/16 : 1), (14 : 1/18 : 0), (14 : 0/18 : 1), etc.  In most cases, such kinds of isomers cannot be separated by the current LC setup, so the combined ID is instead reported.  However, the dominant isomer can be identified by looking at the collision-induced dissociation of tandem MS/MS spectra.

Polyunsaturated Fatty Acid Chains (PUFA)

• Nannochloropsis oculata was found to be a rich source of highly polyunsaturated fatty acids, which included eicosahexaenoic acid, eicosapentaenoic acid, and eicosatetraenoic acid.
• The diacylglycerol backbone for different lipid species is of great interest for PUFAs.

CONCLUSION

• Identification of algae polar lipids required a combination of extraction to isolate the lipid fraction, HPLC to overcome the high dynamic concentration range, ultrahigh resolution FT-ICR MS for unique elemental composition, and tandem MS/MS to identify polar headgroups as well as fatty acid chain lengths and degrees of unsaturation.
• Lipid elemental composition is assigned from accurate mass measurement of the molecular ion and from isotopic fine structure at baseline resolution, possible only with FT-ICR MS.
• Recent improvements in FT-ICR mass resolution and speed of data acquisition, mass calibration and accuracy, and higher magnetic field further extend its reliability and range.

AUTHOR: Steven R. Johnson

CITES:

• Algae Polar Lipids Characterized by Online Liquid Chromatography Coupled with Hybrid Linear Quadrupole Ion Trap/Fourier Transform Ion Cyclotron Resonance Frequency (primary article)
  
• Method for Lipiodomic Analysis
• Stored Waveform Inverse Fourier Transform Axial Excitation
• Space Charge Effects in Fourier Transform Mass Spectrometry - Mass Calibration
• Comparison and interconversion of the two most common frequency-to-mass calibration
• Wikipedia