Method and apparatus for the analysis of molecules using mass spectrometry and optical spectroscopy
A method of analyzing molecules, comprising: generating ions from a sample of molecules; cooling the generated ions below ambient temperature; fragmenting at least some of the cooled ions by irradiating the ions with light at a plurality of different wavelengths (λ) within one or more predetermined spectral intervals; recording a fragment mass spectrum of the fragmented ions comprising a detected signal (I) versus m/z over a predetermined range of m/z values for each of the plurality of different wavelengths (λ), thereby recording a two-dimensional dependency of the detected signal (I) on m/z and irradiation wavelength (λ); and determining from the recorded two-dimensional dependency an identity of at least one of the generated ions and/or relative abundances of different generated ions and thereby determining an identity of at least of one of the molecules and/or relative abundances of different molecules in the sample.
1. A method of analyzing molecules comprising:
causing a sample to flow and subjecting the flowing sample to a separation process whereby different molecules in the flow become separated in time and the concentration of two or more of the molecules in the flow each going through a corresponding local maximum;
generating ions from the sample of molecules to be analyzed;
cooling the generated ions below ambient temperature;
fragmenting at least some of the cooled ions by irradiating the ions with light, wherein the wavelength of the light is scanned over a plurality of different wavelengths within at least two spectral intervals, the at least two spectral intervals each chosen to excite one or more specific molecular bonds of a respective molecule;
recording a fragment mass spectrum for each local maximum, comprising a detected signal (I) versus m/z for a range of m/z values for each of the plurality of different wavelengths (λ), thereby recording a two-dimensional spectrum of the detected signal (I) versus m/z and irradiation wavelength (λ); and
determining from the recorded two-dimensional spectrum an identity and/or abundance of the molecule;
wherein the one or more specific molecular bonds of the molecule comprise one or more of the following:
i. a bond to an isotopic label in an isotopically labeled molecule;
ii. a bond to a functional group or moiety in an organic molecule;
iii. a bond to a functional group in an organic polymer;
iv. a bond in a linker in a cross-linked peptide, or protein, or a complex thereof, or DNA, or RNA;
v. a non-covalent bond in a peptide, protein or a complex thereof.
2. A method according to claim 1 wherein in i. the isotopic label comprises 2 D, 13 C, 15 N, or 18 O, or any combination thereof.
3. A method according to claim 1 wherein in ii. the bond to a functional group or moiety in an organic molecule comprises one of: hydrocarbyls, halogen, oxygen-, nitrogen-, sulfur-, phosphorus-, iron-, selenium-containing groups.
4. A method according to claim 1 wherein in iii. the functional group is a phosphorylation or glycosylation group.
5. A method according to claim 4 wherein the molecule is a peptide, or protein, or DNA, or RNA, or modified peptide, or modified protein, or modified DNA, or modified RNA.
6. A method according to claim 1 wherein in iv. the bond is a disulfide bond or the linker is an artificially introduced linker.
7. A method according to claim 1 wherein in v. the non-covalent bond is a hydrogen bond.
8. A method according to claim 1 wherein in v. the complex is a complex of the peptide or protein with one or more water molecules.
9. A method as claimed in claim 1 wherein the separation process is liquid or gas chromatography and the local maximum is a chromatographic peak.
10. A method as claimed in claim 1 wherein the irradiating the ions comprises sequentially or simultaneously irradiating the ions with light from two or more lasers of different wavelength.
11. A method as claimed in claim 10 wherein one laser has a fixed wavelength and another has a tunable wavelength to allow scanning.
12. A method as claimed in claim 10 wherein the two or more lasers comprise a UV laser and an IR laser.
13. A method as claimed in claim 1 wherein recording the fragment mass spectrum is performed using a linear ion trap mass analyzer, an orbital trap mass analyzer, FT-ICR mass analyzer, or TOF mass analyzer.
14. A method as claimed in claim 1 wherein one or more experimental conditions of the method are selected on the basis of previously acquired data and/or upon fulfillment of one or more predetermined conditions.
15. A method as claimed in claim 1 wherein the two at least two spectral intervals are chosen to excite different specific molecular bonds of the respective molecule.