Method of localizing lipid double bonds

Abstract

A method of mass spectrometry for analyzing lipids and similar biological molecules is disclosed. The lipid molecules may be ionized to form a plurality of lipid parent ions and subjected to photon-induced fragmentation to form a plurality of fragment or product ions. The position of one or more unsaturated bonds in the lipid molecules may be determined by mass analyzing the fragment and product ions and analyzing their intensity profile.

Claims

1. A method of mass spectrometry comprising: ionising lipid molecules to form a plurality of lipid parent ions; subjecting said lipid parent ions to photon-induced fragmentation in order to cause said lipid parent ions to fragment to form a plurality of fragment or product ions; mass analysing said fragment or product ions, wherein subjecting said lipid parent ions to photon-induced fragmentation causes said lipid parent ions to fragment directly or indirectly; and determining the position of one or more unsaturated bonds in said lipid molecules by analysing an intensity profile of fragment or product ions that correspond with cleavage of carbon-carbon bonds from the end of a hydrocarbon chain of the lipid up to the position of an unsaturated bond within said chain, wherein analysing said intensity profile of said fragment or product ions in order to determine the position of one or more unsaturated bonds in said lipid molecules comprises comparing said intensity profile to one or more previous experimentally obtained intensity profiles or comparing said intensity profile to a predicted, calculated or theoretical intensity profile.

2. A method as claimed in claim 1, wherein said fragment or product ions are multiply or substantially doubly charged.

3. A method as claimed in claim 1, wherein the step of subjecting said lipid parent ions to photon-induced fragmentation comprises directing photons emitted from an incoherent light source or non-laser light source onto said lipid parent ions.

4. A method as claimed in claim 1, wherein said lipid parent ions are caused to fragment via photon induced electron detachment.

5. A method as claimed in claim 1, wherein said lipid molecules comprise one or more triglycerols, glycerophospholiids, sphingolipids, fatty acids, glycerolipids or saccharolipids.

6. A method as claimed in claim 1, further comprising confining said lipid parent ions in an ion guide whilst subjecting said lipid parent ions to photon-induced fragmentation.

7. A method as claimed in claim 1, wherein said lipid parent ions comprise a first charge state and wherein said fragment or product ions comprise a second different charge state or comprise a second charge state that is a higher positive charge state or more positive than said first charge state.

8. A method as claimed in claim 1, wherein said lipid parent ions are substantially singly charged.

9. A method as claimed in claim 1, wherein the step of subjecting said lipid parent ions to photon-induced fragmentation causes said lipid parent ions to fragment by photon induced electron detachment, photodissociation or photo-activation.

10. A method as claimed in claim 1, further comprising causing at least some of said lipid parent ions to interact with excited neutral gas molecules or causing at least some of said lipid parent ions to form radical ions and/or metastable ions.

11. A method as claimed in claim 10, wherein said radical ions or metastable ions subsequently dissociate to form said fragment or product ions.

12. A method as claimed in claim 1, wherein the step of subjecting said lipid parent ions to photon-induced fragmentation comprises subjecting said lipid parent ions to ultraviolet radiation.

13. A method as claimed in claim 1, wherein the step of subjecting said lipid parent ions to photon-induced fragmentation comprises directing photons emitted from a vacuum ultraviolet (VUV) discharge lamp, a glow discharge lamp, an ultraviolet lamp, a lamp onto said lipid parent ions.

14. A method as claimed in claim 1, wherein the step of analysing said intensity profile of said fragment or product ions in order to determine the position of one or more bonds in said lipid molecules comprises determining the position of one or more CC double bonds, carbon double bonds or vinyl bonds in said lipid molecules.

15. A method as claimed in claim 1, further comprising subjecting at least some of said lipid parent ions or said plurality of fragment or product ions to supplementary activation to form a plurality of further fragment or product ions and mass analysing said further fragment or product ions.

16. A method as claimed in claim 15, further comprising selecting by mass or using a mass filter one or more of said lipid parent ions or one or more ions derived from said lipid parent ions prior to the step of supplementary activation.

17. A method as claimed in claim 1, further comprising separating at least some of said lipid parent ions, at least some ions derived from said lipid parent ions or at least some fragment or product ions according to ion mobility prior to mass analysis.

18. A mass spectrometer comprising: an ion source arranged and adapted to ionise lipid molecules to form a plurality of lipid parent ions; a photon-induced fragmentation device arranged and adapted to subject said lipid parent ions to photon-induced fragmentation in order to cause said lipid parent ions to fragment to form a plurality of fragment or product ions, wherein subjecting said lipid parent ions to photon-induced fragmentation causes said lipid parent ions to fragment directly or indirectly; a mass analyser arranged and adapted to mass analyse said fragment or product ions; and a control system arranged and adapted to determine the position of one or more unsaturated bonds in said lipid molecules by analysing an intensity profile of fragment or product ions that correspond with cleavage of carbon-carbon bonds from the end of a hydrocarbon chain of the lipid up to the position of an unsaturated bond within said chain, wherein analysing said intensity profile of said fragment or product ions in order to determine the position of one or more unsaturated bonds in said lipid molecules comprises comparing said intensity profile to one or more previous experimentally obtained intensity profiles or comparing said intensity profile to a predicted, calculated or theoretical intensity profile.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Various embodiments together with other arrangements given for illustrative purposes only will now be described, by way of example only, and with reference to the accompanying drawings in which:

(2) FIG. 1 shows a conventional Collisional Induced Dissociation mass spectrum obtained by fragmenting [M+H] parent ions of the phospholipid 1-2-dioleoyl-sn-glycero-3-phosphocholine in the presence of argon buffer gas;

(3) FIG. 2 shows a fragment or product ion mass spectrum according to an embodiment which was obtained by fragmenting [M+H] parent ions of 1-2-dioleoyl-sn-glycero-3-phosphocholine by irradiating the parent ions for approximately 1 second with UV light in the presence of oxygen buffer gas;

(4) FIG. 3 shows in greater detail a region of the fragment or product ion mass spectrum shown in FIG. 2 and shows an intensity profile of characteristic doubly charged fragment or products ion wherein the intensity profile can then be compared against previously acquired or theoretically generated fragment or product ion intensity profiles in order to determine the location of carbon double bonds in the lipid molecules;

(5) FIG. 4 shows a diagram representing the general structure of a lipid with two alkyl chains (chain 1 and chain 2) and with J and K breakable bonds respectively;

(6) FIG. 5 shows a predicted intensity profile of fragment ions with p1j and p2k=0.1;

(7) FIG. 6 shows a predicted intensity profile of fragment ions with p1j and p2k=0.12 for all bonds except the allylic carbon bond; and

(8) FIG. 7 shows a predicted intensity profile if one less CH.sub.2 group is modelled on one of the chains.

DETAILED DESCRIPTION

(9) A conventional approach to mass analysing lipids will first be discussed.

(10) According to a conventional approach lipid molecules are ionised and are then subjected to Collision Induced Dissociation (CID) by fragmenting the ions in the presence of a buffer gas.

(11) FIG. 1 shows a Collision Induced Dissociation mass spectrum obtained by fragmenting [M+H].sup.+ parent ions of 1-2-dioleoyl-sn-glycero-3-phosphocholine (a phospholipid) having a mass to charge ratio of 786.6 in the presence of argon buffer gas. The Collision Induced Dissociation mass spectrum is dominated by a large ion peak corresponding with fragment or product ions having a mass to charge ratio 184. Other less intense fragment or product ions having a mass to charge ratio of 552 are also observed.

(12) FIG. 1 also shows the structure of the lipid molecule and indicates the bond cleavages that result in the formation of the fragment or product ions which are observed in the fragment or product ion spectrum shown in FIG. 1.

(13) No information concerning the position of the unsaturated bonds within the molecule can be determined from the product ion mass spectrum shown in FIG. 1.

(14) A first main embodiment will now be described. The embodiment relates to a method which advantageously enables the position of bonds within a molecule such as a lipid to be determined.

(15) FIG. 2 shows a product ion spectrum of the same lipid parent ions as shown in FIG. 1 but wherein the parent lipid ions were irradiated for approximately 1 second with ultra-violet light in the presence of oxygen buffer gas. The process of irradiating the parent lipid ions with ultraviolet radiation causes the parent lipid ions to fragment. The parent or precursor lipid ions were trapped or confined in an RF confined ion guide and the lipid ions were directly illuminated using a commercial photoionisation lamp (Hamamatsu L10706). The photoionisation lamp emits a broad range of light of wavelength between 120 and 300 nm.

(16) An advantageous feature of an embodiment is that the lipid parent ions may be photo-fragmented using an incoherent ultra-violet lamp rather than a more expensive laser.

(17) The product ion spectrum as shown in FIG. 2 contains the same product or fragment ions having mass to charge ratios of 184 and 552 as are observed in a conventional Collision Induced Dissociation product ion spectrum as shown in FIG. 1. However, importantly, many other singly (and multiply) charged product or fragment ions are also observed.

(18) The combination or intensity pattern of some of the product or fragment ions which are observed in the product ion spectrum obtained according to the techniques described herein allows or enables information such as the location of the unsaturated bonds in the hydrocarbon chains to be determined.

(19) An important feature of the FIG. 2 spectrum is that a significant doubly charged radical precursor cation [M+H].sup.2+ having a mass to charge ratio of 393 is produced. This is in contrast to the conventional Collision Induced Dissociation approach as discussed above with reference to FIG. 1 wherein doubly charged radical cations having a mass to charge ratio of 393 are not observed.

(20) Furthermore, an envelope of doubly charged product or fragment ions is produced which is of particular interest and is marked with an asterisk in FIG. 2.

(21) The doubly charged product or fragment ions correspond with product or fragment ions which correspond with cleavage of CC bonds from the hydrocarbon chains up to the position of the unsaturated bond. This suggests localization of charge due to electron detachment at the double bond.

(22) The envelope of doubly charged product or fragment ions which is marked with an asterisk in FIG. 2 is shown in more detail in FIG. 3.

(23) The doubly charged product or fragment ions which are shown in FIG. 3 are due to loss of CH.sub.2 subunits from the hydrocarbon chains. The intensity profile of the envelope of doubly charged product or fragment ions shown in FIG. 3 is indicative of the position of the double bonds on the hydrocarbon chain and enables the position of the double bonds on the hydrocarbon chain to be determined.

(24) The presence of this envelope of characteristic doubly charged product or fragment ions may be explained by the differences in the mechanisms of dissociation between conventional Collisional Induced Dissociation and a radical ion driven process.

(25) Collisional Induced Dissociation may be termed as being a slow dissociation process. In Collisional Induced Dissociation the energy transferred to the ions via collisions with the target gas has time to be distributed throughout the entire structure of the ion resulting in cleavage of the weakest bonds. In the case of the lipid shown in FIGS. 1 and 2 the probability of seeing losses from the hydrocarbon chain is very low and the CO bond which is cleaved to produce product or fragment ions having a mass to charge ratio 184 has a low bond strength. Accordingly, there is a very high probability of the CO bond breaking and hence ions having a mass to charge ratio of 184 being observed.

(26) In contrast to conventional Collisional Induced Dissociation fragmentation processes, radical ion directed fragmentation may be considered as being a fast process and may result in bonds breaking in a substantially stochastic manner i.e. largely independent of bond strengths.

(27) In addition to examining the intensity envelope of the doubly charged product ions from the alkyl chains, the radical precursor ion and/or higher charged product ions may be further excited by a second downstream fragmentation device such as a collision gas cell with or without isolation using a mass filter.

(28) This increases the yield of informative fragmentation and produces extra informative fragmentation from the hydrocarbon chain which may be used to determine or confirm the probable structure of the hydrocarbon chain.

(29) Two methods of using the intensity profile of the observed doubly charged product or fragment ions from the hydrocarbon chains to elucidate the structure of the lipids are discussed in more detail below.

(30) Method 1

(31) According to a first method a fragment ion spectra library of target lipids of known structure may be produced using the fragmentation method as described above.

(32) Characteristic envelopes of fragment or product ions associated with losses from the hydrocarbon chain which are observed in the library of product or fragment ion mass spectra may then be compared with the envelope of fragment or product ions as produced by an unknown analyte ion.

(33) A probability or score of correlation of the envelope of product or fragment ions within the library to that produced from the analyte may then be produced to indicate the likely structure of the hydrocarbon chain.

(34) Various methods of matching the mass spectra may be used. For example, statistical methods such as principle component analysis (PCA) or probabilistic or Bayesian methods may be used.

(35) Method 2

(36) According to a second method model or library data in-silico may be produced.

(37) The method of assigning structure may comprise: (i) determining from the MS and MS-MS spectra the probable number and length of the hydrocarbon chains and possible number of CC double bonds in the hydrocarbon chains. The type of lipid and nature of the polar head group may then be determined.

(38) The method may further comprise: (ii) determining a set of possible structures for the target lipid for different unsaturated bond positions based on information obtained in step (i).

(39) The method may then comprise: (iii) calculating predicted intensity envelopes for the product or fragment ions from losses from the hydrocarbon chains for the structures proposed in step (ii).

(40) The method may then comprise: (iv) measuring the intensity envelope of product or fragment ions from losses from the hydrocarbon chain produced by photon induced electron detachment.

(41) Finally, the method may further comprise: (v) comparing the measured fragmentation intensity envelope as obtained in step (iv) to the calculated envelopes for the possible subset of structures determined in step (iii) to determine the most probable position of the unsaturated bonds in each hydrocarbon chain.

(42) The mass to charge ratio of the intact lipid and the nature of the polar head group may generally be known. In the example shown in FIG. 2 the product or fragment ions having a mass to charge ratio of 265, in conjunction with molecular ion mass to charge ratio value, indicates the length of the hydrocarbon chains and the total number of double bonds may be calculated.

(43) The total width of the distribution of product or fragment ions also gives an indication of the range of structures which are possible. This narrows the set of calculated values required to fit to the data. In addition, Collision Induced Dissociation data may be used to confirm the basic structure of the lipid.

(44) It has been shown for several molecules that fragmentation via the production of radical ions is substantially stochastic in nature (ETD, ECD). This results in relatively random probability of bond breakage as opposed to more deterministic slow fragmentation processes which generally result in cleavage of the weakest bonds.

(45) This property may be used to construct a simple predictive model of the probability of observing mass losses from doubly charged radical cations due to cleavage of one or more CC bonds.

(46) For example, a lipid with two alkyl chains (chain 1 and chain 2) and with J and K breakable bonds respectively may be considered.

(47) The probability of breaking an individual bond at position j on chain 1=p1j and the probability of breaking an individual bond at position k on chain 2=p2k.

(48) A diagram representing this general structure is shown in FIG. 4.

(49) Each doubly charged fragment ion observed at lower mass to charge ratios than that of the doubly charged radical precursor ion corresponds to the loss of n alkyl groups from chain 1 and or m alkyl groups from chain 2.

(50) The total number of alkyl groups lost is N wherein N=n+m.

(51) The probability of seeing a loss of N alkyl groups Pr(N) is given by:

(52) $\begin{array}{cc}\mathrm{Pr}\left(N\right)=\underset{n+m=N,0nJ,0mK}{.Math.}{P}_{\mathrm{nm}}\mathrm{wherein}:& \left(1\right)\\ P_{\mathrm{nm}}=p1\left(J-n+1\right)p2\left(K-m+1\right)\underset{j=1}{\overset{J-n}{.Math.}}\left(1-p1j\right)\underset{k=1}{\overset{K-m}{.Math.}}\left(1-p2k\right)\mathrm{wherein}:& \left(2\right)\\ p1\left(J+1\right)=1& \left(3\right)\\ p2\left(K+1\right)=1& \left(4\right)\end{array}$

(53) Using this simple model with p1j and p2k=0.1 and using the same lengths and structures of hydrocarbon chains as that shown in FIGS. 2 and 3 gives predicted intensities for the fragment ions as shown in FIG. 5.

(54) The general form of the fragmentation envelope produced is very similar to the form observed in the data indicating that to at least a first approximation the fragmentation is indeed stochastic. Variations from this distribution are due to variations in probability of cleavage at certain bonds related to bond strengths.

(55) For a given lipid structure the relative bond strengths of each of the CC bonds in the hydrocarbon chain may be estimated. Molecular modelling software such as GAUSSIAN may be used to determine the relative bond lengths or bond strengths to produce a value corresponding to the relative probability of a CC bond breaking.

(56) Differences in bond strength are likely to vary particularly in close proximity to a vinyl or unsaturated double bond. It is known that bond strengths in the vicinity of the allylic carbon (adjacent to the double bond) are generally weaker. This fact is exploited in organic synthesis reactions.

(57) Assuming p1j and p2j equal 0.12 for all bonds except for the allylic carbon bond (CC bond next to the double bond) which is given a probability of 0.16 results in a fragmentation envelope as shown in FIG. 6. This has features closer to the profile observed in the data shown in FIGS. 2 and 3.

(58) By refining the relative probabilities in this basic model the form of the fragmentation profile may be approximated. Relatively small changes in the position of the double bond can make significant differences in the predicted fragmentation envelope.

(59) For example, if one less CH.sub.2 group is modelled on one of the chains in the example above, then an intensity envelope as shown in FIG. 7 is predicted. This pattern is indicative of the relative position of the double bond in each chain and may be used to localize the double bonds.

(60) Many permutations of chain length and branching and double bond position may be calculated.

(61) The probability of each bond cleaving may be further refined by examining and fitting predicted data to data from known model compounds with known structure.

(62) In fitting the intensity envelope observed in the data to model data it is important to take into account instrumentation effects which may lead to a bias in the intensity profile with mass to charge ratio and or charge state. For example, the effective duty cycle of ion sampling in the pusher region of an orthogonal Time of Flight mass spectrometer is proportional to the square root of mass to charge ratio. If the ratio of singly and doubly charged product ions is theoretically modelled then the response of the ion detector to ions of differing charge state should optionally also be taken into account.

(63) A second main embodiment will now be described. In this embodiment, the fragment or product ions formed by the methods described herein may be separated by ion mobility prior to mass analysis to produce a two dimensional mass vs. drift time plot.

(64) A suitable apparatus for performing this experiment may comprise an ion source, with precursor ions formed in the ion source being subjected to high energy VUV light in an RF confined ion guide or ion trapping region. The high energy VUV light may cause the precursor ions to undergo electron photo detachment as described above to form a doubly charged radical precursor ion. The trapping region may be an RF only pre-filter region an analytical quadrupole.

(65) The doubly charged radical ion may then optionally be selected using a quadrupole mass filter. Selecting this radical ion may result in an advantageous simplification of the final product ion spectrum as any background ions can be removed.

(66) It is also contemplated that ions may be trapped within the analytical quadrupole and irradiated with VUV light within the quadrupole. Further mass isolation of the radical ion may be achieved by application of a resolving DC potential or by application of suitable supplementary excitation waveforms.

(67) The mass selected radical ion may then be further activated to form further product or fragment ions. For instance, the mass selected radical ion may be accelerated into a collision gas cell to undergo collisional induced dissociation. These product ions may then be accumulated and periodically released into an ion mobility separation device. The product ions are thus separated according to ion mobility prior to their mass analysis e.g. in an orthogonal TOF mass analyser.

(68) Optionally, ions exiting the ion mobility separation device may be activated again, e.g. in a further collision gas cell, to form second generation product ions prior to mass analysis.

(69) Cleavages of the radical precursor ion at several different bond positions (e.g. on different alkyl chains) may give rise to multiple product ions having the same mass to charge value. The probability of producing a particular mass to charge value product ion, considering the different fragmentation pathways available, results in the characteristic product ion intensity envelope described above. These different product ions although indistinguishable by mass alone are significantly different in structure and hence different in interaction cross section so that they can be separated by ion mobility. The ion mobility drift times can be used in addition to the mass values and the intensity profile to provide further structural information to identify the analyte molecule.

(70) The two methods of using the intensity profile discussed above may be extended to use this drift time or interaction cross section information.

(71) For instance, in method 1 the drift time or calibrated cross section values may be stored in the library along with the mass and intensity information. In method 2, estimated cross sections for both precursor and product ions may be calculated in silico and used in addition to the calculated mass and product ion intensity values.

(72) Three dimensional gas phase structures may be calculated for ions formed from this compound. Molecular mechanics and quantum chemistry modelling approaches are commonly employed for this purpose. For instance, software such as Gaussian (www.gaussian.com) is commercially available for these calculations. From these structures it is possible to calculate collision cross sections using software such as MobCal from Indiana University (A. A. Shvartsburg and M. F. Jarrold An Exact Hard Spheres Scattering Model for the Mobilities of Polyatomic Ions, Chem. Phys. Lett. 1996, 261, 86-91).

(73) The effect of long range electronic interactions between ions and polar or polarisable molecules in the IMS drift gas may on apparent collision cross section may also be taken into account with these calculations (Mesleh et al. Structural Information from Ion Mobility Measurements: Effects of the Long-Range Potential, J. Phys. Chem. 1996, 100, 16082-16086).

(74) As part of these calculations it may be necessary to obtain cross sections that have been thermally averaged (e.g. to account for internal movement of the ions in the gas phase).

(75) Where it is not possible to obtain precise theoretical predictions, and a structure does not exist in a library, it may still be possible to correlate patterns in the two-dimensional data with structural details such as the number and positions of double bonds and the number and lengths of lipid chains.

(76) The addition of the second dimension of IMS separation for the product ions produces a three dimensional pattern, mass to charge ratio, intensity and drift time (or collision cross section) which is very specific for the lipid type and the position and number of unsaturated bonds in the ion. Combining the methods described with ion mobility separation increases the confidence in the assignment of the structure of the compound analysed.

(77) It should be noted that reduction of the mass to charge ratio, IMS data to mass to charge ratio and drift time pairs for individual mass to charge ratio peaks is not necessary or in some cases desirable to compare the two three dimensional (mass to charge ratio, intensity and drift time) data to library data or in silico modelled data. Comparing the raw mass to charge ratio drift time image data prior to any feature or component identification or post processing allows features such as IMS peak broadening or tailing or other shape information to be matched to the same features measured or modelled during building of the library data.

(78) Localisation of other groups of molecules containing double bonds within hydrocarbon chains may be investigated by the techniques described above. For example, other embodiments are contemplated wherein the molecules which are analysed comprise hydrocarbons, vegetable oils, fatty acids, fatty aldehydes or ketones.

(79) Other peaks in the spectrum may be used to give information about the length of the hydrocarbon chain of chains and the probable number of double bonds to add to the confidence in assignment. Different fragmentation methods may be combined to give the maximum structural information.

(80) The intensities of fragment ions may be summed or binned within mass to charge ratio regions corresponding to the loss of carbon subunits and different number of hydrogen atoms. This results in the envelope of fragment ions seen for each nominal CH.sub.2 loss appearing as a single intensity value. These intensity values may then be compared with simplified model data comparing the general form of the intensity envelope of the fragment ions to the model data or previously acquired library treated in the same way.

(81) The method of predicting the intensity of fragments may be applied to fragmentation by other radical directed processes such as Electron Transfer Dissociation (ETD), Electron Capture Dissociation (ECD) and metastable atom dissociation (MAD).

(82) Although the present invention has been described with reference to particular embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.