Analysis of the Oligosaccharides and Protein Content of Beer Using Matrix-Assisted Laser
Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI TOFMS)
Elsa Gorre, Ashley Phetsanthad, Jon Soffer, Kevin G. Owens
Department of Chemistry, Drexel University, Philadelphia, PA 19104
Introduction
Results
Results
MALDI TOFMS is becoming an important analysis technique in the brewing industry due to its ability to detect
both the proteins and oligosaccharides present in the beer. Work being done now attempts to use the technique as a
tool for both quality control and for determining beer authenticity.1 Before MALDI can be used as a routine tool,
however, a greater understanding of the effect of sample preparation variables must be obtained. In a MALDI
experiment, sample preparation variables include pretreatment of the sample (such as dilution or solid phase
extraction), choice of matrix and solvent used to dissolve the matrix, the amount of matrix added to the sample , and
sample deposition method. It has already been shown that the quality of the mass spectra obtained is matrix
dependent2; therefore, it is expected that the choice of matrix and solvent will affect the results. Due to the complexity
of the beer samples, pretreatment via solid phase extraction (e.g., using Zip Tips) can also affect the quantity and
type of analytes present in the sample.
Effect of the Matrix
Comparing Oligosaccharide Spectra of Beers Using DHB
Methods
Figure 1: Four beer samples analyzed.
Coors Light beer samples (1μL) were
analyzed using 4μL SA matrix in 3
solvent mixtures:
1. 25ACN/74H2O/1TFA,
2. 50ACN/49H2O/1TFA,
3. 75ACN/24H2O/1TFA
Figure 5: Spectra of CL beer diluted 1:1 with H2O, mixed with 4 different
matrices. From top to bottom, CL in 2,5DHB, CL in CHCA, CL in SA, and CL
in Dithranol. All samples were mixed 4:1 (v/v) matrix to analyte.
Figure 2: MALDI matrices. 4,5
Keeping constant matrix, SA in
50ACN/49H2O/1TFA, different
methods of preparing the analyte
solution were further investigated:
1. Untreated beer
2. 1:1 (v/v) dilution of beer with H2O
3. Concentrated beer solution using
ZipTips
MALDI samples were prepared using
all three analytes preparation methods,
but the ones generating best results
were chosen, shown in Table 1.
Previously it was determined that diluting the beer
sample 1:1 with H2O, yielded better signal compared to
undiluted sample. This method was then carried on as
the analyte preparation method and is investigated using
4 different matrices (DHB, CHCA, SA and Dithranol).
Figure 5 shows the individual spectra obtained with each
matrix. Each spectrum displays peaks believed to be
pertaining to proteins found in Coors Light beer. The first
3 spectra display some of the same peaks, but zooming
in on the spectra shows that SA displays better resolved
peaks compared to CHCA and DHB, especially in the 34kDa region. Out of the four matrices, Dithranol is the
only one that shows a completely different spectrum from
the others. In the Dithranol spectrum, the only visible
peak is around 4kDa, which as explained before it is due
to a fragment from Protein Z. The fragment of Protein Z
is easily missed with the other 3 matrices but on the
other hand shows readily with Dithranol. This interesting
finding supports the claim that understanding sample
preparation is detrimental to understanding your results .
Dithranol was kept as an additional matrix to further
investigate the protein patterns.
Results
Comparing Protein Spectra of Beers Using SA & Dithranol
a).
Table 1: Table displaying the type of analyte
preparation used for each type of matrix.
b).
Table 2: The three matrices used for further investigation of proteins and oligosaccharides in beer
samples. For each matrix, the solvent/solvent system used are reported above, together with the
concentration reported as mg/mL and the volume of matrix added per 1µL of analyte.
TOFMS Instrumental Parameters
All spectra were acquired on a Bruker (Bremen, Germany) Autoflex III MALDI TOF-MS running FlexControl version 3.4 in positive ion
linear mode. The instrument was previously focused and calibrated using a mixture of 5 proteins. Method parameters are as follows:
IS1, IS2 and lens voltages of 19.5kV, 18.25kV and 9.55kV with a PIE delay of 200 or 600ns; 200 shots were collected per spectrum ,
with 50 shots per raster spot in which random walk was activated; the 355nm Nd:YAG laser repetition rate was 100 Hz. Spectra were
analyzed using FlexAnalysis version 3.4.
Figure 8: Stacked oligosaccharide spectra of four beers (Coors Light,
Budweiser, Fegley’s and Flying Fish) in DHB. Spectra were acquired from 14kDa region by mixing 15µL of matrix solution with 1µL of beer sample.
Figure 9: Zoom in oligosaccharide spectrum of Budweiser in DHB.
The zoom in, shows the 7 and 8 oligomer of glucose, which has a
repeat unit of 162Da. On top right corner, calculations are shown of
how to obtain the oligomer information and end group from single
peak.3
DHB matrix was chosen as the best matrix for the oligosaccharide analysis because it is expected to be quite
hydrophilic and experimentally it yielded the best spectra of the matrices investigated. Figure 8 displays the
oligosaccharide spectra of all four beers. At first glance the spectra look very much the same; unfortunately the
intensities cannot be compared from one spectra to another because the samples were deposited via the dry drop
method, and it is known that the method yields heterogeneity in the sample. Note that the oligosaccharide peaks for
Coors Light have an equal intensity of of Na+ and K+ cationized peaks, but the distribution has changed to 1:3
(Na+:K+) for Budweiser. As for the two IPA beers, the K+ peak intensity is much higher in comparison to the Na+ peak.
The reason why this is observed is still under investigation.
Figure 9 shows an expansion of the Budweiser spectrum in Figure 8. In a cluster of two peaks, the first peak
corresponds to a sodium cationized (Na+) peak, while the second peak, separated by 16Da, corresponds to the
potassium cationized (K+) peak. After the oligosaccharide patterns are obtained with DHB matrix, calculations can be
performed to obtain the number of repeat units for a particular oligomer as well as the end groups. Example
calculations are shown for the peak observed at 1175.3Da.
Results
Effect of Solvent System & Analyte Preparation
Figure 3: MALDI spectra of Coors Light in 3 solvent systems. The blue spectrum is of
CL in SA dissolved in 25ACN/74H2O/1TFA of concentration of 2.6mg/mL. The matrix
was mixed in 4:1 (v/v), and 1uL of sample was deposited dry drop on plate for all 3
solutions. The red spectrum is of CL in SA dissolved in 50ACN/49H2O/1TFA with
concentration 20.5mg/mL. The green spectrum is of CL in SA dissolved in
75ACN/24H2O/1TFA with concentration 24.9mg/mL. Two zoom in of the original
spectrum are seen on the top right corner.
Conclusion
Figure 6: a) The top spectrum is of Safbrew WB-06 yeast used to brew beer, and it is analyzed using 2,5DHB as the matrix. Spectra of four beer samples
(Coors Light, Budweiser, Fegley’s and Flying Fish) were analyzed with SA; analyte samples were diluted 1:1 with H2O, and mixed ratio 1:4 (v/v) with SA.
The boxed area of the spectrum is zoom in between the 5 to 11kDa region shown in part b) overlapped spectrum of the four individual spectrum, shown a
closer look at the peaks present in the 5-11kDa region.
Figure 4: Spectra of CL sample, prepared 3 separate ways,
mixed with SA dissolved in 50ACN/49H2O/1TFA. The top
spectrum is using the beer sample as is, while the middle
spectrum is of beer diluted 1:1 with H2O. The bottom spectrum
is of beer sample concentrated down using C4 ZipTips.
The effect of matrix solvent composition and different analyte preparation methods are investigated in Figures 3 and 4. SA was chosen
to study the effect of solvent composition due to its relative high solubility in the mixtures. The solvent mixtures are composed of 1%
trifluoroacetic acid ( TFA) and varying amounts of acetonitrile ( ACN) and water ( H2O ). Figure 3 shows the three spectra of Coors
Light (CL) taken using the different solvent compositions . The three spectra are very similar to one another; and most of the peaks
are present in all three. There is a peak located ~4kDa, which is a fragment of Protein Z.2 This peak is observed as a sharp peak in
the 75ACN mixture , while is not observed at all in the 25ACN mixture. There are only two other peaks present in all three spectra that
have been successfully identified as lipid transfer protein 1 (LTP1) with an average molecular weight of 9694Da, and nonspecific lipid
transfer protein 2 (LTP) with an average molecular weight of 6987Da.3These two proteins can be seen in Figure 3 showing a multiplet
pattern; it has been found that the peaks are 162Da different in mass , suggesting that this is due to the proteins being glycosylated.
From the study of solvent systems, it was concluded that SA dissolved in 50ACN/49H2O/1TFA worked best for the analysis of proteins
because of the better resolution of the peaks as well as higher signal-to-noise ratio obtained. The second focus was on investigating
the effect of analyte pretreatment, such as dilution or extraction via ZipTips , on the signal and total peaks observed in the spectrum.
Figure 4 shows the spectra of CL prepared three ways. All three spectra look similar, although more peaks are observed using
ZipTips, especially in the region of 5kDa. Comparing all three spectra, the spectrum of the analyte diluted 1:1 shows narrower peaks
with less noise. Due to that, diluting the beer samples in a 1:1 ratio with distilled water was chosen as the best analyte preparation
method for the SA matrix.
Figure 7: Spectra of four beers (Coors Light, Budweiser, Fegley’s
and Flying Fish) using Dithranol as matrix. For each beer, C4
ZipTips were used, in which 100µL of beer was aspirated to
concentrate it down, and 10µL of matrix was used to extract the
analytes. In the spectra, the peak corresponding to the fragment of
Protein Z is boxed.
Once the method development was completed, it was
determined that SA matrix worked the best out of the three
matrices (SA, DHB and Dithranol) for protein peaks seen in 3 to
11kDa range. So, using that knowledge, four beer samples were
first diluted and mixed with SA matrix. The spectra of the four
beers are shown in Figure 6, spectrum 2 to 5. Comparing the
four beers to one another, there are some differences in the 3 to
5kDa range. The biggest focus was on comparing the four
beers in the 5 to 11kDa range; due to high intensity of the low
mass peaks, peaks in the higher mass were barely seen in
Figure 6. That is why, a zoom in was done between the 5 to
11kDa region, which scaled the higher mass peaks up making it
easier to compare them. In the part B of Figure 6, the four
spectra of beer were overlapped and unexpected results were
seen for Fegley’s beer. Compared to the other three beers,
Fegley’s does not contain the known protein LTP1. Also, the
unidentified peak at 5kDa is not present in Fegley’s, but it
seems present in the other beers. Expectations were that since
Fegley’s and Flying Fish were both IPA’s, they would have very
similar spectra, but as it was shown in both parts of Figure 6,
that is not the case; they are rather more different compared to
each other rather than to Coors Light and Budweiser. Spectra
of the four beers were also analyzed using Dithranol as the
matrix, and similar to the Dithranol spectrum seen before, the
fragment for Protein Z shows very strongly ~4kDa region. Figure
7 displays each spectrum, and in comparing the four spectra it
seems worthwhile mentioning that the fragment peak is
different. For the two IPA beers, the peak is better resolved,
and the three forms of the peptide can be identified as the
protonated, sodiated and potassiated. The sodiated and
potassiated peaks are not distinguishable in the first two
spectra. The reason as to why that is happening is not known
but will be investigated.
The data presented here are the results of preliminary work done to explore the protein and oligosaccharide
patterns observed in beer. So far it has been shown that different beers have distinct MALDI spectra, which are
thought to be a mixture of analytes coming from the grain, hops and yeast used to brew the beer. At first, method
development was done to identify the matrices that work in producing cleaner spectra (higher signal-to-noise ratio
spectra with better resolved peaks). It was determined that Sinapinic Acid works best for most proteins while
Dithranol was better for only one peptide, which is a fragment of Protein Z. It is still unclear why Dithranol has a
higher affinity for this fragment of Protein Z. Hoteling et al. measured the HPLC retention times for the most common
MALDI matrices, and found that out of the four matrices used here, Dithranol had the longest retention time, which
means that it is more hydrophobic compared to the other matrices. 6 It is believed that the fragment of Protein Z is
hydrophobic and that could explain the higher affinity the Dithranol has toward it.
As seen throughout the poster, beer samples are a mixture of multiple analytes and the identification of all the
peaks is still not yet known. Only a few proteins such LTP 1 and LTP 2, which are both heat resistant proteins found
in barley, are found to be present in the beer sample. The goal is to determine the identity of the other components of
the beer and to determine if there are proteins present from the strain of yeast used in fermenting the beer. In Figure
6a, the first spectrum is of the Safbrew WB-06 dry yeast, and as shown, protein peaks characteristics of the yeast are
visible in the same regions as some peaks observed in the beer. This will be further investigated by brewing beer in
the lab and analyzing each component of the brew individually and then the final product.
References
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