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An Instrumental Technique Evaluation of Organic Structure and Mass Classification by ESI-LTQ/ LIT Mass Spectrometry

Johnathan Harvell

CHEM 431-L01

02 December 2015

Abstract

The purpose of this experiment was to demonstrate and evaluate the efficiency of ESI- LTQ/ LIT MS in the context of mass classification, chemical composition, and structure determination of unknown organic compounds, including proteins. As seen in the data obtained and observations made in this experiment, it was concluded that ESI-LTQ/ LIT MS was a very efficient mass spectrometric instrumentation technique to be used in the context of determining these variables of unknown organic compounds; however, further conclusions of the efficiency of ESI-LTQ/LIT MS in the context of other experimental measurements outside the realm of the variables defined in this experiment could not be made, and further experiments would need to be performed to validate future conclusions.

Introduction

Molecular Mass Spectrometry Theory

Molecular mass spectrometry (MS) is the study of the molecular masses of atoms/molecules, molecular fragments, isotopes, and is a very widely used technique of instrumentation in laboratories across the world; this is primarily based on the technique having the high capability of determining the molecular composition of many unknown organic compounds and corresponding isotopes of organic elements (C, H, O, N, etc). 1 Isotopes are atoms that have a greater mass than the most stable form of element on the periodic table due to the inclusion of a greater number of neutrons inside the atoms nucleus. It is this change in mass by the presence of isotopes and other intermolecular forces that drive the theory of mass spectrometry. MS utilizes a mass spectrum to determine the composition of various samples by graphing the signal strength of a MS detector and the m/z (mass-to-charge) ratio of the molecules found within the sample, where the mass of the molecule is measured in Daltons. 1 By definition, the mass-to-charge ratio is a unit less ratio determined by the division of the molecular mass of a molecular ion and the fundamental charge of the molecular ion. 2 The unit of a Dalton is determined by the mass of the most abundant isotope in the world, 12 C, which leads to the following relationship between Daltons and an Atomic Mass Unit (amu): 2

1 = 1

12

6

= 12 = 12

1 =

1

12

12

6

(1)

(2)

(3)

The m/z allows the separation of molecules in a sample to occur based on a molecular ion’s interaction with a magnetic field within the mass spectrometer. In most mass spectrometers, sample molecular ions are pushed into an electric field, which gives them an initial, specific kinetic energy depending on the ion’s respective mass. This can be further demonstrated by relating the kinetic energy of the molecular ion to the energy of the electric field that the molecular ion is passing through in the following equation:

1 2 = => = √ 2
2

(4)

where (m) is the mass of the molecular ion, (v) is the velocity of the molecular ion, (z) is the fundamental charge of the molecular ion, (e) is the elementary charge of an electron, and (V) is the electric field potential. 1 As the molecular ion is accelerated into the mass spectrometer, the molecular ion will travel perpendicular to a magnetic field that is induced by a constant electric potential placed throughout the path length of the molecular ion. Due to this specific direction of interaction, the magnetic force placed upon the molecular ion during travel can be seen as equivalent to centripetal force of a basic mass, therefore the following relationship can be inferred:

2

= =>

=

(5)

where (B) is the strength of the induced magnetic field, and (r) is the implicit radius of curvature of the path length that the molecular ion is traveling. 1 Since the velocities of equations (4) and (5) correspond to the same molecular mass, these equations can be set equal to each other and used to create the following relationship of the mass-to-charge ratio to the applied electric potential and magnetic forces found in the path length traveled by the molecular ion inside the mass spectrometer: 1

2 2

=

2

(6)

The m/z detected is dependent on the presence of molecular ions and isotopes found within the sample at the point of detection, and is regulated by the process of how ionization or fragmentation occurs; for example, a sample of ethyl benzene may yield the following reaction in a collision with an electron: 3

(7)

As can be seen in equation (7), assuming that the mass of the electron lost is negligible, the radical ion produced in the collision has the exact same nominal mass as ethyl benzene. In a mass spectrometer, the produced ions of such collisions are sorted according to their m/z ratios, therefore, if the charge of the radical ion can be determined, then the nominal mass of the molecule present in the sample can be determined as well. To evaluate the charge of the radical ion, the following equations are used in respect to selected M and M+1 peaks from an obtained mass spectrum:

6 5 2 3 + 6 5 2 3 .+ +

2

=

+1

(8)

= +1

(9)

where (m M ) is the mass of the M peak, (m M+1 ) is the mass of the M+1 peak, (n) is the charge state of the molecular ion, (m p ) is the mass of a proton. 1 For equation (7), the charge of radical ion is +1, with the nominal mass of the ethyl benzene is 106 Da, the m/z peak of 106 will correspond to the presence of the radical ion C 6 H 5 CH 2 CH 3 .+ , therefore can be extrapolated to the identification of ethyl benzene. Although, it is to be noted that many other compounds can have similar nominal masses in relation to ethyl benzene, which can cause further error in data if not identified properly; however, many types of mass spectrometric techniques have been discovered and can be used specifically on different types of samples depending on physical state, quantity, chemical properties, etc.

To further validate a structure of presented in a mass spectrum, the number of rings and double bonds present within a structure can also be calculated by the inference of the elements found within the unknown compound using the following equation:

+ = −

2

+

2

= 1

(10)

where (DB) is the number of double bonds present in a structure, (R) is the number of rings present in a structure, (c) is the number of Group 4A atoms present, (h) is the number of hydrogen/halogen atoms, and (n) is the number of Group 5A atoms present in the unknown compound. 1 It is to be noted that the calculated values produced by equation (10) is a sum of both the double bonds and ring found in the unknown structure, therefore multiple combinations of the double bonds and rings present in the unknown compound can be made. With these multiple combinations, multiple proposed structures of an unknown compound can be illustrated, and will need to be further evaluated by fragmentation techniques to help isolate which proposed structure is the correct one based on the m/z values found in the unknown compound mass spectrum.

The resolution of mass spectrums are important in identifying unknown compounds based on the easily a mass spectrum can be interpreted correctly with the largest signal-to-noise ratio. Resolution is MS is primarily based on the spacing of the masses detected in the mass spectrometer and can be calculated either of the two following equations:

=

=

1

2

(11)

(12)

where (R) is the resolution of the mass spectrum, (m) is the calculated molecular mass of the radical ion in Daltons, (Δm) is the difference in mass between the M and M+1spectral peaks, and (m 1/2 ) is the half height of the M+1 spectral peak. The greater the resolution, the more distinguished each peak on the mass spectrum will be, which allows better interpretation of mass spectral peaks at very low mass difference. This ability in being able to distinguish peaks at a low mass difference allows a better ability to identifying what type of isotopic patterns may be found in a particular sample, thus allowing a more enhanced ability to determining the composition and structure of the sample as well.

Electron Spray Ionization

One particular type of a widely used mass spectrometric technique is that of electron spray ionization (ESI). ESI utilizes the process of electron ionization to where a molecule is collided with an electron or proton to produce molecular ions to be filtered by a mass spectrometer for further analysis. 3 In Figure 1, ESI is performed by allowing a liquid sample to be pumped into a stainless steel

allowing a liquid sample to be pumped into a stainless steel Figure 1: ESI/MS with Quadrupole

Figure 1: ESI/MS with Quadrupole Mass Filter Apparatus Diagram

capillary tube with a

weak flux (typically at a rate ranging from a few nL to μL per minute). 3 As the sample is passing through the capillary, a cylindrical cathode that surrounds the needle point of the capillary at a separation distance of 0.3-2 cm is consistently charged with 3-6 kV of electric potential, which allows the attachment of a charge to the sample as it becomes an aerosol when leaving the capillary. 35 The resulting droplets then began to evaporate, thus allowing the charge density of each droplet to increase in proportion to the size of the evaporating droplet. 35 The charge density steadily increases to a point called the Rayleigh Limit, where the surface tension of the droplet will no longer support the induced charge created by the capillary needle. 35 When this limit is reached, the droplet disassembles into smaller droplets that will tolerate the induced charge, which is commonly called a coulombic explosion. This process is repeated multiple times within the smaller droplets until the capillary solvent has completely evaporated and leaves only the charge molecular ion. 35

Linear Transmission Quadrupole Mass Filtering

To allow a specific molecular ion produced by ESI to be analyzed by the mass spectrometer, a mass separation of the ions must be produced based on the principles of electrostatics and magnetism presented in equation (6). To do so, many scientist use what is called a linear transmission quadrupole (LTQ) mass filter, which utilizes the interactions of molecular ions of different masses and charges in an oscillating electric field. In Figure 2, a diagram demonstrating a LTQ mass filter can be seen. 6 The molecular ions pass into the quadrupole through small circular hole of a metal plate that is determined by the diameter of the ion beam; as the molecular ions pass through the quadrupole, the ions interact with conflicting electromagnetic forces that are induced in each rod of the mass filter. 7

Each pair of metal rods have either an overall positive or negative charge to them, and like-charge rods are positioned directly across from one another. The induced charges of these rods are produced by a direct connection to a circuit that implements both DC and AC current. 7 The DC current allows

a baseline voltage to be maintained throughout the length of the quadrupole to allow the same energy to be applied to all molecular ions being emitted into mass filter; the AC current allows intermediate, sudden changes in voltage of each rod, which allows an instant change in polarity to occur. 7 Due to this change in polarity, an external magnetic force is applied to the molecular ions as they pass through the mass filter, which allows a change in trajectory to occur. This

the mass filter, which allows a change in trajectory to occur. This Figure 2: Transmission Quadrupole

Figure 2: Transmission Quadrupole Apparatus Diagram

change in trajectory is applied continuously throughout the path length of the quadrupole until only one particular massed molecular ion escapes. This selectivity is dependent on the spike in voltage produced by the AC current in the primary quadrupole circuit as demonstrated in equation (6); the higher the spike AC voltage, the smaller the mass of the molecular ion to escape from the mass filter into the detector. 7

Another application of a LTQ is the implementation of selected reaction ion selectivity, which is where a collision occurs to a selected molecular ion from one quadrupole mass filter with an inert gas (such as nitrogen or helium) and the fragments of the collision are then passed through another LTQ mass filter for further molecular ion selection. 3 This technique allows for higher resolution and detection limit of one ng per one mL of sample, which yields detection of sample at the pg level. 1 This technique is highly effective in experiments that are determining very low concentrations of a particular compound in an extremely high quantity of sample. An example of a LTQ apparatus utilizing selected reaction ion selectivity can be seen below in Figure 3: 4

reaction ion selectivity can be seen below in Figure 3: 4 Figure 3: Selected Reaction Ion

Figure 3: Selected Reaction Ion Selectivity LTQ Apparatus Diagram

Quadrupole Linear Ion Trap and Electron Multiplication Detection

With a specific massed molecular ion filtered from the rest of the pumped sample, the ion can then be contained in what is known as a octopole linear ion trap (LIT). 5 A LIT uses a similar rod formation as seen in LTQ to trap molecular ions for further fragmentation and detection. A diagram of a 2D LIT can be seen in Figure 4. 4 As the selected molecular ion exits the mass filter, the rods of the LIT are tuned to the same charge of the molecular ions, which caused repulsion of the molecular ion by the rods. 5 Since the rods are oriented to where they are surrounding the selected molecular ion, the molecular ion becomes stationary, thus being “trapped” by the electrostatic forces applied by the similarly charge rods around the ion. 5 With the ion stationary, fragmentation can be readily performed by the introduction of electrons through a gateway filament or introduction of an inert gas via a chromatography outlet as seen in Figure 4. 3 As

fragmentation occurs and the fragments exit the trap due to momentum given by fragmentation collisions, emitted electrons from the selected molecular ion are funneled into an electron multiplier detector by a conversion dynode as seen in Figure 4. 3 The electron multiplier is laced with Pb at its surface, which provides an electron sea that can be used to amplify the sample signal. As the emitted electrons from the fragmentation collide with the Pb surface, a cascade of electrons eject from one side of the detector, and then hit the surface of the other side of the detector to eject even more electrons; this domino effect of emitted electrons allows better resolution of the sample signal, thus better interpretation of data from the mass spectrum produced by the mass spectrometer.

Experimental Procedure

Mass Confirmation of Multiple Proteins

One 500 μL sample of cytochrome c and another 500 μL sample of myoglobin both in a 70:30 H 2 O:acetonitrile with 0.1% formic acid solvent was directly ejected separately in a Finnigan ESI-LTQ/ LIT mass spectrometer (Thermofisher Scientific, S/N: LTQ10958) at a rate of 25 μL/min. The mass spectrums were produced and analyzed by Thermo Tune Plus computer program (Thermofisher Scientific) as data was accumulated by the mass spectrometer. After data was accumulated, the mass spectrums were used to determine the mass of each protein and were compared to the literature value provided.

Determination of an Unknown Organic Compound Composition and Structure

of an Unknown Organic Compound Composition and Structure Figure 4: Linear Ion Trap Apparatus Diagram A

Figure 4: Linear Ion Trap Apparatus Diagram

A 500 μL sample of an unknown organic compound containing only C, H, N, O in a

66:34 H2O: MeOH solvent was directly injected into Finnigan ESI-LTQ/ LIT mass spectrometer (Thermofisher Scientific, S/N: LTQ10958) at 25μL/min. The mass spectrums were produced and analyzed by Thermo Tune Plus computer program (Thermofisher Scientific) as data was accumulated by the mass spectrometer. Molecular fragmentation was performed by selected reaction ion selectivity by introduction of helium gas in the LTQ and LIT. The resulting mass spectrum was analyzed and interpreted to create a proposed structure and chemical composition of the unknown organic compound given.

Determination of an Unknown Protein Sequence

A 500 μL sample of an unknown three-peptide long protein in a 70:30 H 2 O: acetonitrile

with 0.1% formic acid solvent was directly injected into Finnigan ESI-LTQ/ LIT mass spectrometer (Thermofisher Scientific, S/N: LTQ10958) at a rate of 25 μL/min. The mass spectrums were processed and analyzed by Thermo Tune Plus computer program (Thermofisher

Scientific) as data was accumulated by the mass spectrometer. Molecular fragmentation of the unknown peptide chain was induced by selected reaction ion selectivity by introduction of helium gas in the LTQ and LIT to help identify specific peptide masses found within in the chain, and then were compared to literature mass values of amino acids for confirmation. After the peptides were identified, simulations of fragmentation of the various sequences of the identified peptides were performed using ChemBioDraw Ultra computer program (Perkin Elmer, v. 14.0.0.117, S/N: 216-194259-9384) to isolate the correct sequence of the peptides.

Results and Discussion

Mass Confirmation of Multiple Proteins

In Table 1, the experimental average mass values of both cytochrome C and myoglobin by use of equations (8) and (9) can be seen compared to the respective literature mass values. The resolution of each protein mass spectrum found in Appendix A calculated using equation (11) are shown in Table 1 below as well: 8

Table 1: Experimental Mass Values of Evaluated Protein

Protein

Experimental

Literature Mass

Percent Error

Resolution

 

Mass (Da)

(Da)

(%)

Cytochrome C

12379.8 12400.0

0.16

13983

Myoglobin

17023.9 17000.0

-0.14

12220

In Table 1, it can be seen that there is a low percent error in the experimental masses calculated for both cytochrome C and myoglobin, which infers that the method performed for the mass confirmation of both proteins is accurate to a degree in relation to the literature mass values used for comparison. In Table 1, it can be seen that the resolution of both mass spectrums are relatively high and is supported further by the separation of the spectral peaks found in Appendix A; equation (11) was used instead of equation (12) for the calculation of resolution of each mass spectrum based on the relevance of data obtained in the experiment. The mass spectrums in Appendix A did not include data that demonstrated half-height values, thus the focus of the mass difference between each spectral peak was used for the resolution calculations instead. The ideal mass spectrum profile of a protein can be seen in both mass spectrums, which further infers that the mass spectrometric technique used in this experiment is sufficient to be used to evaluate the mass of each protein evaluated.

Determination of an Unknown Organic Compound Composition and Structure

In Figure 5, the proposed structure of the unknown organic compound evaluated in the experiment can be seen. In Appendix B, the electron and proton ionization spectrums of the unknown organic compound can be seen. Based on the fragmentation found in proton ionization, it was inferred that proton ionization would yield mass spectrums with more spectral peaks, thus allowing a more accurate evaluation of the organic structure found in Figure 5; however, upon determining the experimental molecular mass of the proposed structure, it can be seen that the deprotonated molecular mass of the proposed structure can be seen with an intense mass spectral

peak in the electron ionization mass spectrum in Appendix B of 392 Da. This observation infers that electron ionization allows a better determination of the mass of the unknown organic compound, but does not yield the same fragmentation as seen in proton ionization. In the electron ionization mass spectrum of Appendix B, it is to be noted that the mass spectrum profile similarly resembles a protein mass profile as seen in the mass spectrums of Appendix A. Due to the proposed structure including terminal groups that resemble a model amino acid structure found in proteins, it can be inferred that this similarity in structure is the source of where the mass spectrum profile occurs from.

In knowing that the chemical composition of the unknown organic compound only

contains the elements C, H, N, and O, it was to be inferred that a majority of the mass would be contributed by C based on equation (1-3), thus allowing a calculation of the number of C found in the compound to be performed based on the highest M and M+1 peaks found at 0C in Appendix C. The highest m/z peaks were used in the calculation based on the assumption that the organic compound would not be fragmented at the time of measurement, therefore the highest mass corresponds to the most probably mass of the unknown organic compound. After determining the mass, it was found the mass of unknown organic compound could not be aromatic based on the non-volatile nature of the sample provided; this allowed the assumption that the unknown organic compound must be long carbon chain with attached H, N, and O. With the high electronegativity of O

attached H, N, and O. With the high electronegativity of O Figure 5: Proposed Structure of

Figure 5: Proposed Structure of Unknown Organic Compound

and high number of bonds by N, it was

assumed that O groups would be at certain intervals throughout the chain with N acting as an interval marker in the compound. This is the reason why N is found at equal distances inside the proposed structure, as well as most O groups being found on opposite side of the organic compound based on the steric hindrance of the similar charge found on the O groups.

In using equation (10), it was determined that there was a total of 5 rings/double bonds in the organic structure and, since it was previously established that the unknown organic compound could not be aromatic, an assumption of the a total of 5 double bonds with no rings being present in the unknown compound was made. With this assumption and the fact that an even number of O was determined, it was found that carboxyl groups fit the assumed profile structure of the O groups. This proposed structure is further insinuated based on the protein-like mass spectrum profile found in the electron ionization mass spectrum in Appendix B due to the similar amino acid terminal structures found on the proposed structure in Figure 5.

Determination of an Unknown Protein Sequence

In Figure 6, the proposed unknown protein sequence can be seen demonstrated, while Appendix C demonstrates the mass spectrums collected during the mass measurements of the unknown protein at various collision energies induced by the introduction of helium gas in the

LIT. During the process of determining what peptides were present in the unknown protein given, it was observed that there were repetitive mass spectral peaks at 179, 233, 290, and 308 m/z. With this observation, it could be assumed that the most probable and stable fragmentations were found at this m/z values, therefore extrapolation in the difference between each prominent mass peak could be performed. In protein deformation, it is to be noted that there is a loss of water, which explains the mass difference in some mass spectral peaks found in Appendix C. With the loss of water identified, further inference of the points of fragmentation in the unknown polypeptide could be determined.

According to literature found, the molar masses of Gln, Cys, and Gly are respectively 129 Da, 103 Da, 57 Da (total mass: 289 Da); therefore, the 290 peak corresponds to the protonated combination of these peptides with a loss of water. 9 This inference is further validated by the 308 peak, which corresponds to the entire proposed structure found in Figure 6 without the loss of water. The peak at 179 can be inferred to be the protonated combination Cys and Gly without the loss of water, and the peak at 233 can then be

evaluated to be protonated combination of Gln and Cys with the loss of water. Since Cys is found in all combinations of the fragments present in the mass spectrums found in Appendix C, it was inferred that Cys must be the center of the polypeptide chain, which allowed the final proposed structure of the unknown protein sequence to be determined in Figure 6.

Conclusion

Based on the evidence found in Figures 4-6, Table 1, and Appendices A-C, it can be concluded that the mass spectrometric techniques of ESI, LTQ, and LIT in combination is a very effective way of determining organic structure and composition at a very high resolution. Since the techniques was used in combination, it cannot be inferred that each mass spectrometric technique are equally efficient alone and further experiments would need to be implemented using each separate technique in a similar context of the experiment performed before other conclusions of efficiency can be made; it must also be noted that the efficiency of ESI-LTQ/ LIT MS cannot be assumed to be as great for other experimental measurements made outside the context of this experiment, and more experiments that explore other experimental measurements would need to be performed using this same combination technique before further conclusions can be made.

Acknowledgements

I would like to acknowledge and thank Susannah Miller and Lindsi Durett for their cooperation and participation in the accumulation of mass spectral data in this experiment. I would like to thank Michael Link for the supervision and advice given throughout the time that this experiment was performed. I would also like to thank Colorado State University for allowing

I would also like to thank Colorado State University for allowing Figure 6: Proposed Sequence of

Figure 6: Proposed Sequence of Unknown Protein

our team to perform this experiment using the ESI-LTQ / LIT mass spectrometer found in their Central Instrument Facility.

References

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(5) Joseph Diverdi: Department of Chemistry, Colorado State University.

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Appendix A: Experimental Cytochrome C and Myoglobin Mass Spectrums

A: Experimental Cytochrome C and Myoglobin Mass Spectrums Note: Mass values are represented in kDa for

Note: Mass values are represented in kDa for both Cytochrome C and Myoglobin at a charge of +1.

Myoglobin Mass Spectrums Note: Mass values are represented in kDa for both Cytochrome C and Myoglobin

Appendix B: Unknown Organic Compound ESI Positive/Negative Mode Mass Spectrums

Appendix B: Unknown Organic Compound ESI Positive/Negative Mode Mass Spectrums
Appendix B: Unknown Organic Compound ESI Positive/Negative Mode Mass Spectrums

Appendix C: Unknown Polypeptide Fragmentation Mass Spectrums

Appendix C: Unknown Polypeptide Fragmentation Mass Spectrums
Appendix C: Unknown Polypeptide Fragmentation Mass Spectrums
Appendix C: Unknown Polypeptide Fragmentation Mass Spectrums