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, 12C, which leads to the
following relationship between Daltons and an Atomic Mass Unit (amu):2
1 = 1 (1)
12
6 = 12 = 12 (2)
1
1 = 12 126 (3)

The m/z allows the separation of molecules in a sample to occur based on a molecular
ions 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 ions 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 = => = (4)
2

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
= (6)
2

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
6 5 2 3 + 6 5 2 3.+ + 2 (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:

= (8)
+1

= +1 (9)

where (mM) is the mass of the M peak, (mM+1) is the mass of the M+1 peak, (n) is the charge
state of the molecular ion, (mp) 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 C6H5CH2CH3.+, 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:

+ = + = 1 (10)
2 2
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:

= (11)


= (12)
1
2

where (R) is the resolution of the mass spectrum, (m) is the calculated molecular mass of the
radical ion in Daltons, (m) is the difference in mass between the M and M+1spectral peaks, and
(m1/2) 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
Figure 1: ESI/MS with Quadrupole Mass Filter Apparatus Diagram
into a stainless steel
capillary tube with a
weak flux (typically at a rate ranging from a few nL to L 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 Figure 2: Transmission Quadrupole Apparatus Diagram

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
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

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
Figure 4: Linear Ion Trap Apparatus Diagram
spectrometer.
Experimental Procedure
Mass Confirmation of Multiple Proteins
One 500 L sample of cytochrome c and another 500 L sample of myoglobin both in a
70:30 H2O: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 L/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
A 500 L 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 25L/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 L sample of an unknown three-peptide long protein in a 70:30 H2O: 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 L/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
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
Figure 6: Proposed Sequence of Unknown Protein
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
our team to perform this experiment using the ESI-LTQ / LIT mass spectrometer found in their
Central Instrument Facility.
References
(1) Harris, D., Quantitative Chemical Analysis, 8th ed.; Freeman & Co.: NY; 2010, Chapter
20.
(2) Skoog, D.; Holler, F. J.; Crouch, R. Principles of Instrumental Analysis, 6th ed.;
Thomson Brooks/Cole: CA; 2010; Chapter 11.
(3) Skoog, D.; Holler, F. J.; Crouch, R. Principles of Instrumental Analysis, 6th ed.;
Thomson Brooks/Cole: CA; 2010; Chapter 20.
(4) Van Orden, A. Lecture Notes 10. Canvas: Colorado State University.
https://colostate.instructure.com/courses/14976/pages/lecture-notes-
10?module_item_id=803635
(5) Joseph Diverdi: Department of Chemistry, Colorado State University.
http://sites.chem.colostate.edu/diverdi/C431/experiments/mass%20spectrometry/r
eferences/ESI%20linear%20ion%20trap%20MS%20book.pdf
(6) Definition of Quadrupole Mass Spectrometry. Chemicool.
http://www.chemicool.com/definition/quadrupole_mass_spectrometry.html
(7) Henchman, M.; Steel, C. Understanding the Quadrupole Mass Filter through
Computer Simulation. JChemEd. 1998, 75, 1049-1054.
(8) Fenn, J.B.; Mann, M.; Meng, C.K.; Wong, S.F.; Whitehouse, C. M. Electrospray
Ionization for Mass Spectrometry of Large Biomolecules. Science 1989, 246, 64-
71
(9) Joseph Diverdi: Department of Chemistry, Colorado State University.
http://sites.chem.colostate.edu/diverdi/C431/experiments/mass%20spectrometry/r
eferences/proteins%20and%20peptides%20MS%20book.pdf
Appendix A: Experimental Cytochrome C and Myoglobin Mass Spectrums

Note: Mass values are represented in kDa for both Cytochrome C and Myoglobin at a charge of +1.
Appendix B: Unknown Organic Compound ESI Positive/Negative Mode Mass Spectrums
Appendix C: Unknown Polypeptide Fragmentation Mass Spectrums