analysis of tert-butyldimethylsilyl derivatives of amino

Microsoft Word - GCMS Lab Report 2.docxBenjamin Kagan
Submitted for Chem 201 Lab Partner: Summer Averill TA: Robert Tracy Date lab performed: 4/5/2017 Date report submitted: 4/19/2017
Benjamin Kagan Chem 201 Lab: GCMS date: 4/19/2017
page 1
acids were analyzed by gas-chromatography mass-spectrometry (GC-MS). Samples of bovine
serum albumin (BSA) protein were denatured and hydrolyzed to individual amino acids under
acidic conditions. Derivatization was achieved through the heated reaction of amino acids with
N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide + 1% tert-butyldimethylchlorosilane
(MTBSTFA + 1%tBDMCS). TBDMS protection of the amino acid backbone carboxyl and
amine groups resulted in significant improvement of volatility, which allowed for analysis by
GC-MS. Separation of 15 amino acids was achieved in 18 minutes using a capillary GC with
DB-5 stationary phase, 140 °C starting temperature, and 8 °C/min ramp. A set of three common
ion fragments ([M-tB]+ = M-57, [M-COOtBDMS]+ = M-159, and [M-COtB]+ = M-85) were
observed by mass spectra for 14 of the 15 observed amino acids. In addition, the molecular ion
[M]+ was observed in low intensities for leucine, phenylalanine, aspartate, and lysine. The mass
spectrum of lysine suggested the presence of an intramolecular cyclization reaction to produce a
TBDMS-protected pipecolic acid species. Amino acids not observed included cysteine,
asparagine, glutamine, arginine, and tryptophan. Deuterated amino acid derivatives were
investigated and their elution times were compared to that of the isotopically natural analogue. It
was observed that deuterium substitution resulted in reduced elution time and that there was
positive correlation between the number of deuterium substitutions and the difference in elution
time between deuterated and natural amino acid analogues.
INTRODUCTION
The use of gas-chromatography mass-spectrometry (GC-MS) in protein analysis is
historically a heavily studied area of research due to its excellent capabilities in separating and
page 2
identifying a large number of compounds from a single mixture. GC-MS provides separation of
compounds through both GC and MS mechanisms. Gas chromatography separates compounds
according relative volatility and strength of analyte-column interactions. These factors can be
controlled by modifying experimental parameters as well as column composition. Following
separation by GC, mass spectrometry provides separation of compounds according to mass to
charge ratio (m/z). This allows for the identification of compounds by ion fragmentation patterns
and offers resolution needed to distinguish minor isotopic differences between species.
Fragmentation patterns can be controlled through modification of ionization techniques.
Despite the powerful capabilities that GC-MS offers, significant practical challenges exist
in the analysis of individual amino acids due to the high polarity and large molecular weights of
amino acids, which result in extremely low volatilities. Compounds with low vapor pressures
pose a problem in analysis by GC-MS because injection rates into the GC are limited by the rate
at which sample can be vaporized. As volatility decreases, the time required to completely
vaporize the analyte increases, resulting in decreased resolution due to peak tailing (1). Amino
acids suffer greatly from this phenomenon due to their high polarity and high molecular weights
(2). Fortunately, significant efforts have gone into the development of derivatization methods
that reduce polarity and subsequently improve peak resolution of amino acids in analysis by GC-
MS.
The first implementation of silyl-protecting groups on amino acids for analysis by gas-
chromatograph was reported by Giesecke and co-workers in 1961 with the addition of
trimethylsilyl (TMS) groups to the carboxyl and amine functional groups of aliphatic amino
acids (3). Subsequent reports showed that TMS-derivatization could be achieved with all 20
common amino acids (4). Although TMS-derivatization provided adequate reduction in
page 3
volatility, challenges stemming from a lack of derivative stability limited the application of
TMS-derivatization in routine amino acid analysis (5). Efforts to resolve this stability issue led to
the introduction of tert-butyl(dimethylsilyl) (TBDMS) as an alternative silyl protecting group to
TMS (6). This derivatization process, outlined in Scheme 1, was achieved by the reaction of
amino acids with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA). It was
observed that TBDMS-derivatized amino acids were significantly more stable than their TMS
counterparts. Further benefits of TBDMS-derivatization included protection of side-chains
containing active protic groups (hydroxyl, amine, sulfhydryl) (7).
(Scheme 1)
EXPERIMENTAL
Theory. The derivatization of amino acids with TBDMS protecting groups serves to
greatly improve the volatility of amino acids by minimizing the polarity of the backbone
hydroxyl and amine groups. This process occurs through a single alkylsilylation step in which
MTBSTFA undergoes nucleophilic attack at silicon by the hydroxyl and amine groups of the
hydrolyzed amino acid. N-methyl(trifluoroacetylamide) is lost as a result of this reaction. In
addition to protection of the backbone hydroxyl and amine groups, amino acids with active
H3N OH
page 4
protic R-groups such as Ser, Thr, His, Lys, and Tyr undergo additional TBDMS silylation at
these positions.
Column chromatography is a separations technique that is used to isolate compounds
according their interactions with the stationary phase and mobile phase of a column. Compounds
that interact strongly with the stationary phase elute at a slower rate compared to compounds that
possess greater affinity for the mobile phase, therefore producing separation. In gas
chromatography (GC), the mobile phase is a gas and the stationary phase is often a liquid.
Modern capillary GC columns utilize a thin film liquid stationary phase, which can be specified
as either polar or non-polar (1). In this experiment, a polar stationary phase was used consisting
of a silicon oil with 5 % of its substituents as phenyl groups (DB-5). Therefore, compounds with
greater polarity would be expected to elute slower than nonpolar compounds.
Quadrupole ion-trap (QIT) mass spectrometers separate compounds according to their
mass to charge ratio (m/z). For singly charged compounds, this means that m/z values represent
the molecular weight of the particular ion. QIT mass spectrometers function by generating a 3D
quadrupolar field via an oscillating electric field that is used to store ions in three-dimensions.
This field is generated between two hemispherical plates and it is modulated by adjusting electric
potential to control the m/z of ions that are held in the field (8).
Analysis of compounds by mass spectrometry requires ionization of the analyte in order
to separate species by their mass to charge ratio (m/z). Electron ionization is a hard-ionization
technique, which produces three commonly observed ion fragments during the analysis of
TBDMS-protected amino acids (7). These ions include the [M-tB]+ (M-57), [M-COOtBDMS]+
(M-159), and [M-COtB]+ (M-85) fragments (Figure 4). In addition to these commonly observed
page 5
fragments, the molecular ion [M]+ is also sometimes observed, however peak intensity is usually
significantly lower than the fragment ions (7).
Materials. A total of three amino acid samples were used to generate GC-MS samples.
Hydrolyzed bovine serum albumin (BSA) in 6 M HCl (4.06 mM Leu) and a four amino acid
mixture (Ala, Leu, Phe, Lys) in water (3.7 mM Leu) were obtained from the UVM Department
of Chemistry. A uniformly deuteratred (2H) algal amino acid mixture (98%+ 2H) was obtained
from Cambridge Isotopes Laboratory. HPLC-grade water (3/12/15), acetonitrile, 1 M acetic acid
(3/11/13, TJV), and 3 M ammonium hydroxide (3/11/17, TJV) were obtained from the UVM
Department of Chemistry. Ion exchange resin in water (AG 50W-X8) was obtained from Bio-
Rad Laboratories. Regis Technologies Inc. provided N-methyl-N-(tert-butyldimethylsilyl)-
trifluoroacetamide + 1% tert-butyldimethylchlorosilane (MTBSTFA + 1%tBDMCS).
Instrumentation. A Varian Saturn 2100T ion trap gas chromatograph-mass spectrometer
with a Varian 3900 GC and CP-8400 autosampler was used to analyze amino acid samples. A
capillary GC column with a polar stationary phase consisting of silicon oil with 5 % substitution
of phenyl groups (DB-5) was used.
Procedures. A total of four unique TBDMS-derivatized amino acid solutions were
prepared for analysis by GC-MS. The samples were analyzed in replicate and concentrations
were generated relative to leucine so that each sample contained approximately 200 nmol of
leucine. A four amino acid standard mixture (Ala, Leu, Phe, Lys) was prepared by adding 50 µL
of a stock aqueous solution (4.06 mM Leu) to a 2 mL screw cap vial. A uniformly deuterated
(2H) algal amino acid mixture was prepared by adding 50 µL of stock deuterated amino acid
solution (3.7 mM Leu) to a 2 mL screw cap vial. A second set of deuterated (2H) algal amino
acid samples were prepared for the BSA D-AA sample.
page 6
Preparation of the two BSA-containing samples required the use of a cation exchange
column in order isolate the amino acids from the acid hydrolysate. The acid hydrolysate (6M
HCl) was used to denature the protein sample and cleave peptide bonds to produce individual
amino acids. The two ion exchange columns were prepared by adding 1 mL of cation exchange
resin (AG 50W-X8) to the column followed by a rinse with 4 mL of HPLC-grade water. A BSA
hydrolysate solution was prepared by combining a 150 µL sample of BSA hydrolysate (2.7 mM
Leu) with 2 mL of 1 M acetic acid. To each column, 150 µL of the hydrolysate solution were
added, the acid eluate was allowed to drain, and the columns were rinsed with HPLC-grade
water (3 x 2 mL). Amino acids were removed from the column by addition of 2 mL of 3 M
ammonium hydroxide to each column. The resulting eluent from one column was collected and
transferred to two new screw cap reaction vials. The eluent from the second column was added
to one set of the previously prepared deuterated amino acid sample vials to generate the
combination BSA D-AA sample.
Solvent was removed from vials using a stream of nitrogen gas in the N-Evap apparatus.
To each vial, 100 µL of a 1:1 mix of MTBSTFA and acetonitrile was added and the vials were
sealed. The vials were placed on a vortex shaker and heated at 110 °C for 30 minutes. The vials
were allowed to cool to temperature before being stored in a refrigerator for one week. After
warming to room temperature, the samples were transferred to GC vials for analysis.
Samples were analyzed by a Varian Saturn GC-MS using electron ionization (EI) and a
scan range of 50-550 Da. Specific experimental parameters included a helium flow rate of
1mL/min, an injector temperature of 250 °C, an initial GC temperature of 140 °C with a 2 minute
hold, and a 8 °C/min GC ramp to 300 °C with a 5 minute hold. Total ion current (TIC)
chromatographs were collected for the four amino acid mixture, the BSA sample, and the BSA
page 7
D-AA combination sample. Neither replicates of the uniformly deuterated (2H) sample produced
a useful TIC. Mass spectra for each amino acid were collected and fragments were identified
according to mass to charge. The BSA D-AA combination sample was further analyzed using an
ion selection trace to compare the ratio of natural (1H) to deuterated (2H) amino acids and to
observe the shift in elution time due to the isotopic substitution.
Data Analysis. Ion fragment intensities tabulated in Tables 1, 2, and 3 were calculated
with Equation 1 where m/z intensities of individual fragments were divided by the sum of the
intensity of all identified fragments.
% ion intensity = intensity ion fragments
(1)
Equations 2 and 3 were used to determine the expected m/z values for deuterated amino
acids. Equation 2 allowed for determination of the expected number of substituted deuterium
atoms where n was the number of aliphatic and aromatic protons in the R-group of the natural
amino acid.
The resulting molecular weights of deuterated fragments were determined by adding the
number of substituted deuterium atoms to the molecular weight of the natural ion fragment
(Equation 3). The molecular weight of the natural ion fragments were previously tabulated (9).
MW of H fragments = MW of H fragment + (1 + n)(1 g/ H>?> (3)
The relative abundance of deuterated and natural amino acids in the BSA D-AA sample
were determined by Equations 4 and 5 respectively, utilizing intensities from the individual ion
select traces.
page 8
Abundance of H> amino acid = H ion select intensity>
H? ion select intensity + H> ion select intensity (4)
Abundance of H? amino acid = H ion select intensity?
H? ion select intensity + H> ion select intensity (5)
The difference in elution time (EFGHIJK, mins) was determined by comparing the elution
time of natural amino acid t(1H) (mins) to that of its deuterated analogue t(2H) (mins). (Equation
6)
RESULTS
The TIC chromatograph of the TBDMS-derivatized four amino acid sample (Ala, Leu,
Phe, Lys) produced a clean set of four peaks corresponding to each amino acid (Figure 1). Using
a starting temperature of 140 °C, alanine was observed to elute first at 5.754 mins followed by
leucine at 7.760 mins, phenylalanine at 12.533 mins, and lysine at 15.658 mins. Mass spectra
with labeled ion fragments for each amino acid are displayed in Figures 1-1 through 1-4. The
observed ion fragments and their relative intensities are reported in Table 2. Alanine, leucine,
and phenylalanine produced [M-tB]+, [M-COOtBDMS]+, and [M-COtB]+ ion fragments, which
are commonly observed for TBDMS-derivatized amino acids. However, the molecular ion [M]+
was not observed for alanine, leucine, or phenylalanine. The mass spectrum of lysine showed the
presence of a cyclized species as evidenced by peaks at 300.3 m/z, 198.2 m/z, and 272.2 m/z
corresponding to [M-tB]+, [M-COOtBDMS]+, and [M-COtB]+ fragments of a cyclized species.
Evidence of linear lysine was observed by the molecular ion [M]+ at 488.5 m/z and the [M-tB]+
fragment at 431.3 m/z.
page 9
Analysis of the BSA sample by GC-MS allowed for the identification of 15 amino acids
(Figure 2). Experimental parameters remained unchanged from the four amino acid sample,
resulting in consistent elution times between the common amino acids of the two samples (Ala,
Leu, Phe, Lys). The observed order of elution for the BSA sample was alanine (5.779 mins),
glycine (6.047 mins), valine (7.263 mins), leucine (7.733 mins), isoleucine (8.190 mins), proline
(8.653 mins), methionine (11.147 mins), serine (11.388 mins), threonine (11.752 mins),
phenylalanine (12.558 mins), aspartate (13.274 mins), glutamate (14.549 mins), lysine (15.669
mins), histidine (16.780 mins), and tyrosine (18.176 mins). Several unidentified peaks were
observed in the TIC but a lack of useful ion fragments prevented assignment of these peaks.
Individual ion fragments for each amino acid were identified by the mass spectra (Figures
2-1 through 2-15) and the relative intensities of the peaks were reported in Table 2. Ten of the 15
observed amino acids: Ala, Val, Ile, Pro, Met, Ser, Thr, Glu, His, Tyr; produced three ion
fragments: [M-tB]+, [M-COOtBDMS]+, and [M-COtB]+. The mass spectrum of glycine
contained [M-COOtBDMS]+ and [M-COtB]+ fragments, but lacked the [M-tB]+ fragment.
Leucine, phenylalanine, and aspartate were characterized by all three fragment ions as well as the
molecular ion [M]+, however the molecular ion was observed with relatively low intensity. As
with the four amino acid standard, lysine was observed in both linear and cyclized
conformations, appearing as two and three unique ions respectively.
The BSA D-AA combination sample provided insight into the effects of deuterium-
substitution on elution time in a gas chromatograph. Experimental parameters were unchanged
from the standard BSA experiment, which used a 140 °C starting temperature and an 8 °C/min
ramp. The TIC of the BSA D-AA sample is presented in Figure 3 and specific elution times are
tabulated in Table 3. As expected, the observed order of elution was consistent with the standard
page 10
BSA experiment and the same 15 amino acids were observed. In addition, the mass spectra of the
natural amino acids (Figures 3-1b through 3-15b) matched those previously collected from the
pure BSA sample. Deuterated samples were observed to elute before their isotopically natural
analogues, however their presence was not obvious by the TIC. Figures 3-2c, 3-3c, 3-4c, 3-5c,
and 3-8c provide an expanded view of the TIC for individual amino acids, demonstrating the
lack of resolving power that would be required to visual the individual isotopes by TIC.
Regardless, deuterated amino acids were quantified by scanning the TIC upfield of natural signal
until the deuterated sample was observed in the mass spectrum.
Deuterated versions of all 15 amino acids were observed, as evidenced by highly similar
fragmentation patterns to those of the natural isotope. Figures 3-1a through 3-15a present the
mass spectra of the deuterated analogous and elution times are tabulated in Table 3. Ions
corresponding to the deuterated samples were uniformly heavier for all fragments of a particular
amino acid. Aside from a few exceptions, nearly all ion fragments observed for the natural
samples were also observed for the deuterated samples. Exceptions included the molecular ion
[M]+ of leucine, phenylalanine, and aspartate; which appeared as low intensity ions in the natural
amino acids.
To compare the difference in elution time between deuterated and natural amino acids,
ion select traces were used to generate chromatograms corresponding to single m/z values.
Traces were generated corresponding to the base peaks of the natural and deuterated versions of
glycine (Figure 3-2c), valine (Figure 3-3c), leucine (Figure 3-4c), isoleucine (Figure 3-5c), and
serine (Figure 3-8c). It was observed that amino acids with greater deuterium substitution tended
to produce the greatest shift in elution time. For example, when comparing the aliphatic R-
groups of valine and leucine, leucine exhibited an elution shift of 0.063 mins compared to 0.052
page 11
mins for valine. It was also observed that between amino acids of similar deuterium substitution,
amino acids containing TBDMS-substituted R-groups tended to display reduced shifts in elution
time as evidenced by the elution shift of 0.048 for threonine compared to 0.052 for valine. Table
4 lists the difference in elution time (mins) between deuterated and natural versions of each
amino acid.
In the BSA D-AA sample, the relative abundance of deuterated and natural forms of a
select number of amino acids were calculated by comparing the chromatogram intensity of the
ion select traces. For all amino acids investigated (Gly, Val, Leu, Ile, and Ser), the deuterated
form made up just a minor part of the sample. Of the five, glycine-d was the most abundant of
with a fraction of 0.12, followed by serine-d (0.07), isoleucine-d (0.05), valine-d (0.03), and
leucine-d (0.02).
DISCUSSION
Although GC-MS serves as an excellent analytical technique for the analysis of protein
composition, the highly polar nature of individual amino acids presents significant practical
challenges in obtaining high resolution spectra. The challenge with highly polar compounds is
due to their inherently low volatility, which makes it difficult to achieve rapid injection rates into
the gas chromatograph upon heating. This results in poor separation of species due to peak
broadening and severely limits resolution. This problem can be avoided by increasing the
volatility of individual amino acids through a derivatization process. The addition of a sterically
bulky group such as TBDMS to the highly polar hydroxyl and amine groups of an amino acid
significantly increases volatility and allows for the collection of high resolution spectra.
This derivatization process is characterized by a single alkylsilylation step in which a
TBDMS groups are added at the carboxyl and amino positions of each amino acid (Scheme 1)
page 12
(7). This process is initiated by the nucleophilic attack at silicon in MTBSTFA to generate a five-
coordinate transition-state around silicon, followed by the loss of N-methyl-trifluoracetamide to
produce the TBDMS-protected species. In all amino acids, both the amine and hydroxyl groups
were sufficiently basic to perform the nucleophilic attack of MTBSTFA and TBDMS protection
was observed at both groups. In addition, amino acids possessing hydroxyl and amino-containing
R-groups gained additional TBDMS protecting groups on these groups. This derivatization
process is catalyzed by 1% TBDMCS (5).
In the BSA sample, 15 of the 20 common amino acids were identified in the TIC and
corresponding mass spectra were obtained. The amino acids that were not observed included
cysteine, asparagine, glutamine, arginine, and tryptophan. The absence of cysteine in the TIC is
explained by the presence of disulfide bonds between sulfhydryl groups of individual cysteine
residues. Unfortunately, these linkages prevent efficient derivatization of sulfhydryl groups and
therefore volatility cannot be improved to allow for gas chromatograph elution. Reduction of
these disulfide linkages with the addition of a reducing agent would significantly improve
derivatization and likely allow for analysis by GC-MS (10). As with cysteine, derivatization of
arginine poses a challenge, however the difficulty is due to sterics rather than inter-residue
linkages. The side chain of arginine is characterized by a three carbon aliphatic chain and a
terminal guanidine group. This terminal guanidine group poses a challenge since the close
proximity of nitrogen atoms prevents silylation by TBDMS at each nitrogen due to steric
limitations (10). As a result, derivatization of arginine requires significantly more energy relative
to the other amino acids. Asparagine and glutamine were not observed since they are hydrolyzed
to aspartate and glutamate upon protein denaturation by acid hydrolysis due to the loss of their
page 13
amide groups (5). Finally, the absence of tryptophan can be explained by the lack of significant
amounts of tryptophan in BSA protein.
The order of elution of TBDMS-derivatized amino acids was dependent on two main
factors: size and polarity. It was observed that amino acids with nonpolar, aliphatic side-chains
eluted first, followed by more polar species and that amino acids with large, aromatic side-chains
eluted last. This trend was consistent with previous findings that utilized columns with polar
stationary phases such as DB-5 (5). It is important to recognize that elution time is dependent on
both volatility and analyte-column interactions. Species with greater volatility elute earlier
because they are introduced to the column before species with lower volatility. If two species are
equally volatile, separation is purely due to interactions with the column. In this experiment, the
polar stationary phase of the gas-chromatograph interacted more strongly with polar species
compared to non-polar species. For this reason, alanine eluted prior to glycine due the lower
polarity of alanine despite alanine having a greater molar mass subsequently lower vapor
pressure than glycine.
The relationship between polarity and volatility on elution time was further explored by
measuring the effect of substituting deuterium for R-group and a-hydrogen atoms in the
investigated amino acids. For all natural amino acids observed in the BSA sample, deuterated
analogues were observed to elute before their isotopically natural counterparts and this trend was
consistent with previous findings (11). As previously discussed, elution time is influenced both
by analyte volatility as well as analyte-column interactions. In comparison to the overall weight
of the TBDMS-protected amino acid, substitution of deuterium had little effect on volatility since
the resulting mass change (2-10 m/z depending on the amino acid) was not large enough to
significantly affect vapor pressure. In fact, if only volatility was considered, it would be
page 14
predicted that deuterated amino acids would elute after their natural analogues. Because
experimental observations showed faster elution times for deuterated amino acids, it was
concluded that the difference in analyte-column interactions was responsible for the change in
elution time.
The faster elution times of deuterated species was due to decreased Van-der-Waals
interactions with the polar, DB-5 stationary phase (12). There was a positive correlation between
the degree of deuterium substitution and the difference in elution time. Evidence of this trend
included the increase in Dtelution as deuterium substitution increased from 4 to 8 to 10 in aliphatic
amino acids (alanine = 0.039 mins, valine = 0.052 mins, isoleucine = 0.069 mins) (Table 4).
Further support for the importance of analyte-column interactions in elution time included the
observation of a shielding effect on deuterium by TBDMS-protected R-groups. This shielding
effect reduced the impact of deuterium substitution because the sterically bulky TBDMS group
reduced visibility of deuterium to the column. Evidence of this shielding effect was observed by
the difference in Dtelution for amino acids containing similar degrees of deuterium substitution but
varying degrees of TBDMS-protection. For example, threonine and valine share similar R-group
geometries but threonine contains a TBDMS protecting group on its terminal hydroxyl group
whereas valine possesses two methyl groups. The shielding effect of TBDMS is demonstrated by
the lower Dtelution value of 0.048 mins for threonine compared to 0.052 mins for valine.
For most of the amino acids analyzed, a common set of three ion fragments were
observed by mass spectra. These fragments included the [M-tB]+, [M-COOtBDMS]+, and [M-
COtB]+ ions (Figure 4). The [M-tB]+ ion was a result of the loss of the terminal tert-butyl group
from the TBDMS, producing an ion 58 m/z lighter than the molecular ion [M]+. Generation of
the [M-COOtBDMS]+ fragment occurred through elimination of the carboxyl and TBDMS
page 15
protecting group resulting in a loss of 159 m/z. The mechanism to form the [M-COtB]+ fragment
involved a molecular rearrangement in which dimethylsilane was transferred to the carboxyl
carbon with subsequent elimination of COtB resulting in a loss of 85 m/z relative to [M]+.
In addition to the commonly identified ion fragments, consistent signals at 73 m/z and
302 m/z were observed in the mass spectra for multiple amino acids. Due to their presence in
spectra corresponding to range of amino acids, it was hypothesized that these common signals
corresponded to degradation products of the TBDMS-derivatized amino acids. Therefore, it was
suggested that trimethylsilane (TMS) produced the signal at 73 m/z and that the 302 m/z peak
was caused by the TBDMS-derivatized amino acid backbone (no R-group).
The mass spectrum of lysine produced particularly interesting results. It was predicted
that lysine would produce strong signals at 431.25 m/z, 329.25 m/z, and 403.26 m/z
corresponding to the [M-tB]+, [M-COOtBDMS]+, and [M-COtB]+ ion fragments commonly
observed for other amino acids. Unlike the majority of amino acids, the only predicted fragment
that experimentally observed was the [M-tB]+ ion. The mass spectrum contained three other
significant peaks at 300.3 m/z, 198.3 m/z, and 272.4 m/z. It was hypothesized that these signals
were due to the presence of a product of an intramolecular cyclization of TBDMS-protected
lysine (Scheme 2). This species, referred to as lysine (cyclized) in this report, is a TBDMS-
protected form of pipecolic acid (MW = 129.157 g/mol). This cyclization process has been
heavily studied in biological settings since it is a natural step in the metabolism of lysine. It has
been shown that mechanism of this intramolecular cyclization involves the nucleophilic attack
and subsequent loss of the a-amino group (13). (Scheme 2)
page 16
(Scheme 2)
In this experiment, TBDMS-derivatized amino acids were analyzed by GC-MS. A total
of 15 of the 20 common amino acids were successfully separated and common fragmentation
pattern consisting of three ions were observed ([M-tB]+ = M-57, [M-COOtBDMS]+ = M-159,
and [M-COtB]+ = M-85). The molecular ion [M]+ was detected for a limited number of amino
acids. It was observed that volatility was largely responsible for the difference in elution time
between different amino acids as evidenced by the grouping of aliphatic, polar, and aromatic
amino acids in the order of elution. Comparison of deuterated amino acids with their natural
analogues demonstrated the effect of polarity on elution time as evidenced by the faster elution
time of the less polar deuterated analogues. Unfortunately, peaks corresponding to the deuterated
analogues could not be resolved in the TIC chromatogram due to the extremely low fraction of
deuterated amino acid relative to the natural isotope (Table 5). Although deuterated peaks were
not resolved by TIC, evidence of an isotopic shift was clearly observed by ion select traces
corresponding to the base peaks of the isotopically natural and deuterated species.
H NO
page 17
LITERATURE CITED 1. Matthews, D. E. (2017) Chem 201 GC Lecture.
2. Martin, N. (1965) Mass spectra of twenty-two free amino acids. Stanford Univ.
3. Ruhlmann, K., and Giesecke, W. (1961) Gas chromatography of silylated amino acids.
Angew. Chem. 73, 113
4. Gehrke, C. W., Nakamoto, H., and Zumwalt, R. W. (1969) Gas-liquid chromatography of
protein amino acid trimethylsilyl derivatives. Journal of Chromatography A 45, 24-51
5. Goh, C. J., Craven, K. G., Lepock, J. R., and Dumbroff, E. B. (1987) Analysis of all
protein amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid
chromatography. Anal. Biochem. 163, 175-181
6. Mawhinney, T. P., and Madson, M. A. (1982) N-Methyl-N-(tert-
butyldimethylsilyl)trifluoroacetamide and related N-tert-butyldimethylsilyl amides as
protective silyl donors. J. Org. Chem. 47, 3336-3339
7. Mawhinney, T. P., Robinett, R. S. R., Atalay, A., and Madson, M. A. (1986) Analysis of
amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography
and mass spectrometry. J. Chromatogr. 358, 231-242
8. de Hoffmann, E., and Stroobant, V. (2007) Mass Spectrometry Principles and
Applications, John Wiley & Sons
9. Matthews, D. E. (2006) tBDMS-Derivative EI Ionization.
10. Chaves das Neves, H. J., and Vasconcelos, A. M. P. (1987) Capillary gas
chromatography of amino acids, including asparagine and glutamine: sensitive gas
chromatographic-mass spectrometric and selected ion monitoring gas chromatographic-
page 18
Chromatogr. 392, 249-258
11. Zolotarev, Y. A., Zaitsev, D. A., Lubnin, M. Y., Tatur, V. Y., and Myasoyedov, N. F.
(1988) Isotopic effects in chromatomass-spectrometry of deuterium-substituted amino
acids. International Journal of Radiation Applications and Instrumentation. Part A.
Applied Radiation and Isotopes 39, 619
12. Meier-Augenstein, W. (2002) Stable isotope analysis of fatty acids by gas
chromatography–isotope ratio mass spectrometry. Analytica Chimica Acta 465, 63-79
13. Rothstein, M., and Miller, L. L. (1954) Loss of the α-amino group in lysine metabolism
to form pipecolic acid. J. Am. Chem. Soc. 76, 1459
page 19
Table 1. Ion fragments measured by GC-MS of four TBDMS-derivatized amino acid standards using a starting temperature of 140 °C and 8 °C/min ramp. Ion fragments were identified by mass to charge ratio (m/z) and the relative intensity (%). A total of four ions were observed including the molecular ion (M+) as well as three fragmented ions [M-tB]+, [M-COOtBDMS]+, and [M-COtB]+. Elution time (mins) was determined by maximum intensity of the GC trace. Tabulated expected ion fragments for TBDMS-protected amino acids (9). M+ [M-tB]+ [M-
COOtBDMS]+ [M-COtB]+
Amino Acid m/z % m/z % m/z % m/z %
5.754 Ala - - 260.0 21.2 158.2 27.2 232.2 51.5 7.760 Leu - - 302.2 18.6 200.2 40.2 274.3 41.2 12.533 Phe - - 336.2 29.4 234.3 19.6 308.2 50.9 15.658 Lys (linear) 488.5 7.1 431.3 5.3 - - - -
Lys (cyclized) - - 300.3 37.5 198.2 34.4 272.2 15.6 Table 2. Ion fragments measured by GC-MS of a TBDMS-derivatized hydrolyzed bovine serum albmin (BSA) sample using a starting temperature of 140 °C and 8 °C/min ramp. Ion fragments were identified by mass to charge ratio (m/z) and the relative intensity (%). A total of four ions were observed including the molecular ion (M+) as well as three fragmented ions [M-tB]+, [M- COOtBDMS]+, and [M-COtB]+. Elution time (mins) was determined by maximum intensity of the GC trace. Tabulated expected ion fragments for TBDMS-protected amino acids (9). M+ [M-tB]+ [M-
5.779 Ala - - 260.2 18.8 158.2 30.4 232.2 50.7 6.047 Gly - - - - 246.0 30.9 218.0 69.1 7.263 Val - - 288.2 16.3 186.3 40.6 260.3 43.0 7.773 Leu 360.2 2.4 302.2 19.7 200.2 35.9 274.3 42.0 8.190 Ile - - 302.2 23.9 200.2 37.6 274.3 38.5 8.653 Pro - - 286.2 12.1 184.2 56.7 258.2 32.2 11.147 Met - - 320.2 20.5 218.2 25.6 292.2 53.8 11.388 Ser - - 390.2 34.5 288.4 15.0 362.5 50.5 11.752 Thr - - 376.5 24.8 303.3 46.4 404.2 28.8 12.558 Phe 394.2 7.0 336.2 27.4 234.2 17.9 308.2 47.6 13.274 Asp 476.5 7.2 418.3 43.5 316.5 15.5 390.4 33.8 14.549 Glu - - 432.5 76.1 330.6 18.8 404.5 5.1 15.669 Lys (linear) 488.5 7.8 431.3 5.9 - - - -
Lys (cyclized) - - 300.3 42.9 198.3 16.1 272.4 27.3 16.780 His - - 442.3 58.7 340.3 34.8 414.4 6.5 18.176 Tyr - - 466.5 52.2 364.4 14.4 438.5 33.3
page 20
Table 3. Ion fragments measured by GC-MS of a TBDMS-derivatized hydrolyzed bovine serum albmin (BSA) and uniformly deuterated (2H) amino acid sample using a starting temperature of 140 °C and 8 °C/min ramp. Ion fragments were identified by mass to charge ratio (m/z) and the relative intensity (%). A total of four ions were observed including the molecular ion (M+) as well as three fragmented ions [M-tB]+, [M-COOtBDMS]+, and [M-COtB]+. Elution time (mins) was determined by maximum intensity of the GC trace. Tabulated expected ion fragments for TBDMS-protected amino acids (9). M+ [M-tB]+ [M-
5.729 Ala-d - - 264.2 22.2 152.2 28.7 236.2 49.0 5.768 Ala - - 260.2 21.1 158.2 28.8 232.2 50.1 5.999 Gly-d - - - - 248.1 17.6 220.2 82.4 6.040 Gly - - - - 246.2 34.0 218.2 66.0 7.202 Val-d - - 296.2 16.7 194.2 40.3 268.2 43.0 7.254 Val - - 288.2 18.6 186.3 39.3 260.3 42.1 7.710 Leu-d - - 312.2 21.0 210.2 43.2 284.3 35.8 7.773 Leu 360.2 0.2 302.2 18.5 200.2 37.1 274.3 44.2 8.112 Ile-d - - 312.2 17.2 210.2 40.0 284.3 42.8 8.181 Ile - - 302.2 23.5 200.2 37.1 274.3 39.4 5.594 Pro-d - - 293.3 9.6 191.2 55.3 265.2 35.1 8.649 Pro - - 286.2 12.4 184.2 53.6 258.2 34.0 11.080 Met-d - - 329.2 17.9 226.3 22.2 300.3 59.9 11.140 Met - - 320.2 18.0 218.2 22.5 292.2 59.5 11.344 Ser-d - - 393.3 29.1 302.3 26.3 365.3 44.6 11.383 Ser - - 390.2 36.8 288.4 16.2 362.3 47.0 11.705 Thr-d - - 381.3 26.1 308.3 27.3 409.3 46.6 11.753 Thr - - 376.5 24.5 303.3 46.2 404.2 29.2 12.504 Phe-d - - 344.2 19.2 242.3 21.0 316.3 59.8 12.552 Phe 394.2 13.4 336.2 43.7 234.2 35.1 308.2 7.7 13.229 Asp-d - - 421.2 40.1 319.3 21.7 393.3 38.1 13.275 Asp 476.5 3.7 418.3 42.3 316.5 14.9 390.4 39.1 14.504 Glu-d - - 437.4 75.1 335.3 21.2 409.6 3.7 14.549 Glu - - 432.5 74.7 330.5 19.5 404.5 5.8 15.603 Lys-d (linear) 497.6 12.1 440.4 9.2 - - - -
Lys-d (cyclic) - - 309.2 18.3 207.9 40.8 281.4 19.5 15.667 Lys (linear) 488.5 6.5 431.3 5.0 - - - -
Lys (cyclic) - - 300.3 40.0 198.3 31.8 272.4 16.7 16.728 His-d - - 449.5 66.7 347.5 33.3 - - 16.769 His - - 442.5 59.4 340.5 33.8 414.5 6.7 18.139 Tyr-d - - 473.5 51.9 371.4 15.1 445.5 33.0 18.186 Tyr - - 466.5 47.8 364.4 19.5 438.5 32.7
page 21
Table 4. Difference in elution time (Dtelution, mins) between deuterated and natural amino acids in the BSA D-AA sample. Deuterated versions were observed to before the natural analogue. Amino Acid Dtelution (mins) Ala 0.039 Gly 0.041 Val 0.052 Leu 0.063 Ile 0.069 Pro 0.055 Met 0.060 Ser 0.039 Thr 0.048 Phe 0.048 Asp 0.046 Glu 0.045 Lys 0.064 His 0.041 Tyr 0.047
Table 5. Percent deuterated (2H) amino acid in the BSA – 2H-AA sample. Fractions of deuterated and 1H amino acids were determined by comparing intensity of the ion select trace for the respective base peak (m/z). Amino Acid D (2H) base
peak (m/z) D (2H) fraction 1H base
peak (m/z)
1H fraction
Gly 220.2 0.12 218.2 0.88 Val 268.2 0.03 260.3 0.97 Leu 284.3 0.02 274.3 0.98 Ile 284.3 0.05 274.3 0.95 Ser 365.3 0.07 362.5 0.93
page 22
Figure 1. Total ion current (TIC) chromatogram of the TBDMS-derivatized four amino acid sample (Ala, Leu, Phe, Lys) using a 140 °C starting temperature and an 8 °C/min ramp. Elution time (mins) for each amino acid are reported in Table 1.
page 23
Figure 1-1. MS spectrum of TBDMS-derivatized alanine. Three ion fragments were observed as reported in Table 1.
Figure 1-2. MS spectrum of TBDMS-derivatized leucine. Three ion fragments were observed as reported in Table 1.
page 24
Figure 1-3. MS spectrum of TBDMS-derivatized phenylalanine. Three ion fragments were observed as reported in Table 1.
Figure 1-4. MS spectrum of TBDMS derivatized lysine. Two ion fragments (blue) corresponding to the linear form and three ion fragments (green) corresponding to the cyclized form were observed as reported in Table 1.
page 25
Figure 2. Total ion current (TIC) chromatogram of TBDMS-derivatized bovine serum albumin (BSA) using a 140 °C starting temperature and an 8 °C/min ramp. Elution time (mins) for the 15 observed amino acids are reported in Table 2.
page 26
Figure 2-1. MS spectrum of TBDMS-derivatized alanine. Three ion fragments were observed as reported in Table 2.
Figure 2-2. MS spectrum of TBDMS-derivatized glycine. Two ion fragments were observed as reported in Table 2.
page 27
Figure 2-3. MS spectrum of TBDMS-derivatized valine. Three ion fragments were observed as reported in Table 2.
Figure 2-4. MS spectrum of TBDMS-derivatized leucine. Three ion fragments were observed as reported in Table 2.
page 28
Figure 2-5. MS spectrum of TBDMS-derivatized isoleucine. Three ion fragments were observed as reported in Table 2.
Figure 2-6. MS spectrum of TBDMS-derivatized proline. Three ion fragments were observed as reported in Table 2.
page 29
Figure 2-7. MS spectrum of TBDMS-derivatized methionine. Three ion fragments were observed as reported in Table 2.
Figure 2-8. MS spectrum of TBDMS-derivatized serine. Three ion fragments were observed as reported in Table 2.
page 30
Figure 2-9. MS spectrum of TBDMS-derivatized threonine. Three ion fragments were observed as reported in Table 2.
Figure 2-10. MS spectrum of TBDMS-derivatized phenylalanine. Four ion fragments were observed as reported in Table 2.
page 31
Figure 2-11. MS spectrum of TBDMS-derivatized aspartate. Four ion fragments were observed as reported in Table 2.
Figure 2-12. MS spectrum of TBDMS-derivatized glutamate. Three ion fragments were observed as reported in Table 2.
page 32
Figure 2-13. MS spectrum of TBDMS-derivatized lysine. Two ion fragments (blue) corresponding to the linear form and three ion fragments (green) corresponding to the cyclized form were observed as reported in Table 2.
Figure 2-14. MS spectrum of TBDMS-derivatized histidine. Three ion fragments were observed as reported in Table 2.
page 33
Figure 2-15. MS spectrum of TBDMS-derivatized tyrosine. Three ion fragments were observed as reported in Table 2.
page 34
Figure 3. Total ion current (TIC) chromatogram of TBDMS-derivatized bovine serum albumin (BSA) and uniformly deuterated (2H) amino acid sample using a 140 °C starting temperature and an 8 °C/min ramp. Elution time (mins) for the 15 observed 1H and 2H amino acids are reported in Table 3.
page 35
Figure 3-1a. MS spectrum of TBDMS-derivatized deuterated (2H) alanine. Three ion fragments were observed as reported in Table 3.
Figure 3-1b. MS spectrum of TBDMS-derivatized alanine. Three ion fragments were observed as reported in Table 3.
page 36
Figure 3-2a. MS spectrum of TBDMS-derivatized deuterated (2H) glycine. Two ion fragments were observed as reported in Table 3.
Figure 3-2b. MS spectrum of TBDMS-derivatized glycine. Three ion fragments were observed as reported in Table 3.
page 37
Figure 3-2c. Expanded TIC trace of glycine (red) in the BSA D-AA sample. Ion select traces for natural glycine (green trace) and deuterated (2H) glycine (orange trace) show separation of the isotopes.
page 38
Figure 3-3a. MS spectrum of TBDMS-derivatized deuterated (2H) valine. Three ion fragments were observed as reported in Table 3.
Figure 3-3b. MS spectrum of TBDMS-derivatized valine. Three ion fragments were observed as reported in Table 3.
page 39
Figure 3-3c. Expanded TIC trace of valine (red) in the BSA D-AA sample. Ion select traces for natural valine (green trace) and deuterated (2H) valine (orange trace) show separation of the isotopes.
page 40
Figure 3-4a. MS spectrum of TBDMS-derivatized deuterated (2H) leucine. Three ion fragments were observed as reported in Table 3.
Figure 3-4b. MS spectrum of TBDMS-derivatized leucine. Three ion fragments were observed as reported in Table 3.
page 41
Figure 3-4c. Expanded TIC trace of leucine (red) in the BSA D-AA sample. Ion select traces for natural leucine (green trace) and deuterated (2H) leucine (orange trace) show separation of the isotopes.
Leucine-d m/z 284.3
Leucine m/z 274.3
page 42
Figure 3-5a. MS spectrum of TBDMS-derivatized deuterated (2H) isoleucine. Three ion fragments were observed as reported in Table 3.
Figure 3-5b. MS spectrum of TBDMS-derivatized isoleucine. Three ion fragments were observed as reported in Table 3.
page 43
Figure 3-5c. Expanded TIC trace of isoleucine (red) in the BSA D-AA sample. Ion select traces for natural isoleucine (green trace) and deuterated (2H) isoleucine (orange trace) show separation of the isotopes.
page 44
Figure 3-6a. MS spectrum of TBDMS-derivatized deuterated (2H) proline. Three ion fragments were observed as reported in Table 3.
Figure 3-6b. MS spectrum of TBDMS-derivatized proline. Three ion fragments were observed as reported in Table 3.
page 45
Figure 3-7a. MS spectrum of TBDMS-derivatized deuterated (2H) methionine. Three ion fragments were observed as reported in Table 3.
Figure 3-7b. MS spectrum of TBDMS-derivatized methionine. Three ion fragments were observed as reported in Table 3.
page 46
Figure 3-8a. MS spectrum of TBDMS-derivatized deuterated (2H) serine. Three ion fragments were observed as reported in Table 3.
Figure 3-8b. MS spectrum of TBDMS-derivatized serine. Three ion fragments were observed as reported in Table 3.
page 47
Figure 3-8c. Expanded TIC trace of serine (red) in the BSA D-AA sample. Ion select traces for natural serine (green trace) and deuterated (2H) serine (orange trace) show separation of the isotopes.
page 48
Figure 3-9a. MS spectrum of TBDMS-derivatized deuterated (2H) threonine. Three ion fragments were observed as reported in Table 3.
Figure 3-9b. MS spectrum of TBDMS-derivatized threonine. Three ion fragments were observed as reported in Table 3.
page 49
Figure 3-10b. MS spectrum of TBDMS-derivatized phenylalanine. Four ion fragments were observed as reported in Table 3.
page 50
Figure 3-11a. MS spectrum of TBDMS-derivatized deuterated (2H) aspartate. Three ion fragments were observed as reported in Table 3.
Figure 3-11b. MS spectrum of TBDMS-derivatized aspartate. Four ion fragments were observed as reported in Table 3.
page 51
Figure 3-12a. MS spectrum of TBDMS-derivatized deuterated (2H) glutamate. Three ion fragments were observed as reported in Table 3.
Figure 3-12b. MS spectrum of TBDMS-derivatized glutamate. Three ion fragments were observed as reported in Table 3.
page 52
Figure 3-13a. MS spectrum of TBDMS-derivatized deuterated (2H) lysine. Two ion fragments (blue) corresponding to the linear form and three ion fragments (green) corresponding to the cyclized form were observed as reported in Table 3.
Figure 3-13b. MS spectrum of TBDMS-derivatized lysine. Two ion fragments (blue) corresponding to the linear form and three ion fragments (green) corresponding to the cyclized form were observed as reported in Table 3.
page 53
Figure 3-14a. MS spectrum of TBDMS-derivatized deuterated (2H) histidine. Two ion fragments were observed as reported in Table 3.
Figure 3-14b. MS spectrum of TBDMS-derivatized histidine. Three ion fragments were observed as reported in Table 3.
page 54
Figure 3-15a. MS spectrum of TBDMS-derivatized deuterated (2H) tyrosine. Three ion fragments were observed as reported in Table 3.
Figure 3-15b. MS spectrum of TBDMS-derivatized tyrosine. Three ion fragments were observed as reported in Table 3.
page 55
Figure 4. Common ion fragments of TBDMS-protected amino acids observed by GC-MS.
H NO
O Chemical Formula: C5H9O•

analysis of tert-butyldimethylsilyl derivatives of amino

Documents