Impact of un-polymerized acrylamide monomer residues onto protein identification by MALDI TOF MS

Article abstract of DOI:10.2478/s11532-012-0024-3

Polyacrylamide gel electrophoresis is a powerful tool for protein mixture separation frequently used in proteomic studies. This article describes the problems which can arise during the identification process of separated proteins by matrix assisted laser desorption/ ionization mass spectrometry. Presence of residual acrylamide monomers in the peptide samples leads to a significant peptide signal suppression. In the case of protein prohibitin, isolated from the MCF 7 breast cancer cell line, the peptide signal suppression was observed in 50% of all detected peptides. Versita Sp. z o.o.

Full text of DOI:10.2478/s11532-012-0024-3

Cent. Eur. J. Chem. • 10(4) • 2012 • 1073-1078
DOI: 10.2478/s11532-012-0024-3
Central European Journal of Chemistry
Impact of un-polymerized acrylamide
monomer residues onto protein identification
by MALDI TOF MS
Research Article
Ivan Talian1, Veronika Kováčová1, Marián Petrovič1, Ján Sabo1
1Department of Medical and Clinical Biophysics,
Pavol Jozef Šafárik University 040 11 Košice, Slovakia
Received 30 November 2011; Accepted 12 January 2012
Polyacrylamide gel electrophoresis is a powerful tool for protein mixture separation frequently used in proteomic studies. This article
describes the problems which can arise during the identification process of separated proteins by matrix assisted laser desorption/
ionization mass spectrometry. Presence of residual acrylamide monomers in the peptide samples leads to a significant peptide signal
suppression. In the case of protein prohibitin, isolated from the MCF 7 breast cancer cell line, the peptide signal suppression was
observed in 50% of all detected peptides.
Abstract:
Keywords: MALDI • Acrylamide • Peptide mass fingerprinting • Gel electrophoresis
© Versita Sp. z o.o.
1. Introduction
α,β - unsaturated carbonylic compounds is a well-
characterized chemical reaction [17]. Since protein
Polyacrylamide gel electrophoresis (PAGE) is one of identification after PAGE is mostly performed via mass
the most used separation technique for protein mixture spectrometry,especiallybyMALDITOF[18,19],formation
analysisintheproteomicstudies[1,2].Thepolymerization of adducts during PAGE is not desirable. Proteins that
is rarely quantitative and the polymer usually contains were modified by these adducts have different mobility in
some un-polymerized acrylamide molecules [3,4]. comparison with unmodified proteins during the PAGE.
Chiari and co-workers calculated that for 5%T gel The reason for this phenomenon is the fact that the adduct
(20×20cm×1mm) is the concentration of unreacted free changes the proteins overall charge and eventually its
acrylamideabout30-60mM[5-7].Acrylamide,withrespect size. Tertiary amines – used in solutions of electrolytes
to its chemical properties, is able to react with various by PAGE – change their pH value due to the influence
types of chemical compounds. During the electrophoretic of adducts with acrylamide. Proteins, e.g. bovine
separation acrylamide can react with free sulfhydryl serum albumin and cytochrome c showed a change in
groups in proteins and form cysteine-acrylamide adducts electrophoretic mobility in PAGE after the interaction
[8]. In the proteomic analysis the relevant reactions of with acrylamide monomer under alkaline conditions [20].
acrylamide with primary and secondary amines and In MALDI MS technique, acrylamide adducts increases
eventually with tertiary amines (the last mentioned create the probability of prior ionization of acrylamide instead
buffer solution of electrolyte within PAGE separation) can of peptide ionization (peptide signal suppression) or the
occur [9]. The elimination of amines adducts formation is measured masses shift to a higher m/z due to these
especially important in peptide mass fingerprinting (PMF). adducts. This leads to problematic protein identification
Acrylamide can covalently bind proteins by reacting with and increases the number of false positive identifications.
the amino group [10,11]. For a long time there has been The most likely reaction of un-polymerized acrylamide is
a well-known reaction mechanism of carbamides with with the cysteine [21]. Sulphilic bond of cysteine is prone
amines. Nucleophilic addition of ammonia, primary and to carbamidoethylation. For example Barber and co-
secondary amines [12-14], and amino acids [15,16] to workers observed adducts of acrylamide with cysteine
* E-mail: ivan.talian@upjs.sk
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Impact of un-polymerized acrylamide monomer residues
onto protein identification by MALDI TOF MS
on the peptides of digested protein (recombinant NSF
protein) where the peptide masses were shifted to higher
m/z values. The reaction of cysteine was in this case
carbamidoethylation (mass difference 71 m/z) despite
expected carbamidomethylation (mass difference
57 m/z) [22]. The other amino acids underlying possible
carbamidoehtylation are serine, valine and glutamine.
Recent results indicate that also glycine is able to create
adducts of various types and it can lead to serious
complications concerning the evaluation of mass
spectra [23].
Thearticledescribesproblemsinproteinidentification
by PMF with MALDI TOF caused by un-polymerized
acrylamide residues presented in the digested protein
sample. The influence of acrylamide on peptide
ionization and following peptide mass fingerprinting is
discussed.
2.2.2. UV control of acrylamide polymerization
reaction
UV absorption spectra were recorded on a double
beam Shimadzu UV 1800 spectrometer (Wavelength
range 190 to 1100 nm, Spectral bandwidth, Wavelength
accuracy 0.1 nm), using a 1cm path length.
2.2.3. Two-dimensional electrophoresis and protein
spot analysis
Nuclear proteins were diluted in a chaotropic 2-D
rehydration/sample buffer from BioRad kit. 125 μL of
diluted proteins (1.23 mg mL-1) were loaded into IPG
strips by in-gel passive rehydration strips overnight.
Afterwards isoelectric focusing of the proteins was
conducted at 20ºC with a current limit of max. 50 μA per
strip in linear mode from 250 V to 4000 V until reaching
20000 V hrs in a PROTEAN IEF cell from Bio-Rad
(USA). The IPG strips were subsequently transferred
to an equilibration buffer (6 mol dm-3 urea, 2% SDS,
0.375 mol dm-3 Tris HCl, pH=8.8, 20% glycerol,
0.130 mol dm-3 DTT), 2 mL per strip, followed with
equilibration buffer (6 mol dm-3 urea, 2% SDS,
2. Experimental procedure
2.1. Materials
Cytochrome c and acetonitrile (ACN) were purchased 0.375 mol dm-3 Tris HCl, pH=8.8, 20% glycerol,
from Sigma Aldrich (St. Louis, Mo, USA), Tris base, 0.135 mol dm-3 iodoacetamide). SDS-PAGE was
ammonium persulfate, TEMED, 0.5% agarose, Tris- performed on 12.5% SDS-polyacrylamide gel using
glycine-SDS buffer, 30% acrylamide/bis solution, Mini PROTEAN Tetra Cell apparatus (Bio-Rad
coomassie brilliant blue R-250 destaining solution were Laboratories, USA). Strips were fixed in place with
purchased from Bio-Rad (Hercules, CA), ammonium an agarose overlay. Electrophoresis was carried out
bicarbonate(NH4HCO3)wasfromApplichem(Darmstadt, at 200 V for approximately 50 min until the dye
Germany), trifluoroacetic acid (TFA) was from Acros- (bromophenol blue) front reached the end of
Organics (New Jersey, USA), proteomics grade purity the gel. The running buffer (0.025 mol dm-3 Tris,
trypsin was purchased from Promega (Medison, USA), 0.192 mol dm-3 glycin, 0.1% SDS) was not cooled.
ultra-pure water with current resistance of more than The staining procedure was performed using Coomassie
18.2 mΩ was used for preparation of the electrophoresis brilliant blue (for 1 hour) and afterwards the gels were
buffers and digestion solutions (Millipore, Bedford, MA, washed 3x with proteomic grade water. The stained
USA).
gels were scanned using Calibrated Densitometer
GS – 800 and data were analyzed using the PDQuest
software (Bio-Rad Laboratories (USA). Selected protein
spots were manually cut.
2.2. Methods
2.2.1. MCF 7 nuclear proteins isolation
The source of cytoplasmic protein was the cell line from
breast cancer, from American Type Culture Collection 2.2.4. Protein digestion
(ATCC, USA). Cells were cultured in DMEM with the The gel pieces were transferred to 500 µL tubes and
addition of FBS, Penicillin and Streptomycin at 37ºC in washed with 200 µL of 50% ACN/25 mM ammonium
medium containing 5% CO2 and 95% humidity. Cells bicarbonate buffer for gel decoloration. Supernatant
were collected at 80-90% confluence using trypsin and was discarded and the gel pieces were washed once
ice-cooled PBS. The cells were washed 3 times with with 100 µL 100% ACN. After a few minutes the gel
cooled PBS. Pellet was formed by centrifugation at pieces turned white and ACN was removed. The gel
2500 g and stored at -80ºC. The number of cells in the pieces were dried in the air. Dried gel pieces were
pellet was approximately 2×106. The cell lysis was made rehydrated in 25 µL of 25 mM ammonium bicarbonate
according to the instruction manual from the ReadyPrep containing 12.5 µg trypsin. After 60 minutes on ice, the
Protein Extraction Kit (Cytoplasmic/Nuclear) from Bio- non-absorbed trypsin solution was discarded. 20 µL
Rad Laboratories (USA).
of 25 mM ammonium bicarbonate was added to the
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I. Talian et al.
gel pieces to prevent the gel from drying out and the
–
Homo sapiens (human), global modifications
samples were incubated at 37ºC for at last 12 h. To stop Carbamidomethyl (C), variable modificat ions Oxidation
the digestion process 5 μL of 5% TFA was added and (M), enzyme Trypsin, the number of maximum missed
the samples were shaken out for 15 min. The samples cleavages 1, mass error tolerance 0.1 Da (MS).
were separated with a spinning process and supernatant
was transferred to 500-µL tube. Peptides were extracted
twice with 20 µL of 50% ACN/0.1% TFA and afterwards
combined again.
3. Results and discussion
The PAGE resolution and reproducibility is strongly
2.2.5. MALDI TOF MS
influenced by the gel preparation conditions. The
polymerization can be easily controlled by UV
absorbance due to the fact that the absorbing double
bonds are during polymerization eliminated. The reaction
was controlled at the wavelength of 260 nm (Fig. 1).
Although the reaction has exponential behavior (the
UV absorbance decreases with the increasing reaction
time) and the most of the monomers have reacted
already after approximately 1 h the monomer residues
still presented in the gel strongly influence not only the
protein mixture separation, but also their identification.
The resolution and the reproducibility of the 2D PAGE
is decreasing with the rising concentration of acrylamide
monomer residues. As an example, the image of 2D
PAGE separation of nuclear proteins isolated from the
MCF 7 cell line is shown in Fig. 2.
1 μL of samples were mixed with 2 μL of MALDI matrix
α-cyano-4-hydroxycinnaminic acid (4 mg mL-1 in
acetonitrile/methanol/0.1% TFA in water, 84/14/2 v/v/v)
and 1 μL was spotted on a MALDI target and allowed to
dry. Samples were analysed on a mass spectrometer
UltrafleXtreme MALDI TOF/TOF (Bruker Daltonik,
Germany). Spectra were acquired in the reflection
mode in the range of 700 – 3500 Da. Mass calibration
was performed externally with PepCal standard. The
data were evaluated using the Mascot algorithm (Matrix
Science Ltd., UK). Mass spectra and tandem mass
spectra of peptides were compared against NCBIr
database. The search parameter used was: taxonomy
Fig. 2a shows the separation on the gel prepared
with one hour of solidification time and 2b with 24 hours
respectively. On the gel 2a we can see the unresolved
spots. This leads to the problems connected with the
spot cutting and the number of identified proteins
decreases. The reproducibility of such prepared gels is
also rather poor. For instance the protein HNRF marked
1 or the 60 kDa heat shock protein marked 2 are in the
gel 2a unresolved from its variants, showed almost as
one unresolved line. Meanwhile on the gel 2b we can
see the distinct spots. Except for the resolution and
separation problems the protein identification utilizing
MALDI TOF is also influenced. The conditions during
Figure 1. The acrylamide polymerization process controlled by UV
absorbance at the wavelength of 260 nm.
Figure 2. The image of 2D PAGE separation of nuclear proteins isolated from the MCF 7 cell line, (a) gel solidification time of 1 hour and
(b) a solidification time of 24 hours.
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Impact of un-polymerized acrylamide monomer residues
onto protein identification by MALDI TOF MS
the ionization process are crucial. As an example, and the acquired mass spectra are at least in one order
the MALDI identification of cytochrome C (horse) was of magnitude more intensive. This combination ensures
performed after the gel electrophoresis on the gels also a more accurate peptide mass determination and
prepared with different solidification times. The selected so protein identification with minimal false positive
solidification times were 1, 3, 6, 12 and 24 hours. This matches. The protein prohibitin from the MCF 7 cell
time scale should ensure minimal un-reacted acrylamide was chosen as a model protein for the peptide mass
presented in the gel after polymerization. The number fingerprinting after 2D SDS-PAGE. In Fig. 3 we can see
of matched peptides and their intensity in the MALDI the comparison of prohibition PMF spectrum from the
spectrum were compared. The results are listed in the two gels with different solidification time (1 h and 24 h).
Table 1.
The number of matched peptides was 5 in the 1 h gel
From Table 1 it is clear that the gel solidification and 10 in the 24 h gel. The sequence coverage raised in
time/concentration of un-reacted acrylamide can this case from 16% up to 41.5% and the Mascot score
considerably improve the identification score of the from 68 to 153. Notice that especially heavier peptide
separated proteins. Due to more matched peptides masses are missing (mass 1791.7 m/z do not belong
a better sequence coverage is achieved. Also the MS to peptide masses of protein prohibition). However it
spectrum acquisition is much easier; less laser power should be mentioned that also few lighter peptides were
leadstobettersignaltonoiseratio, bettermassresolution not detected in the 1 hour gel. The comparison of these
MS spectra points out on the ionization problems of
Table 1. The number of matched peptides for cytochrome C in PMF
proteins related with acrylamide monomer residues from
the gel. The possible explanation is the surface-protein
interaction between the peptide and the un-polymerized
acrylamide monomer during the desorption event. In
the extreme case (high concentration of un-reacted
acrylamide, in this case the gel solidification time was
only 30 minutes), the polymerization of acrylamide
induced by the laser pulse can be observed. The free
acrylamide monomer residues show polymeric behavior
(Fig. 4).
MALDI analysis after PAGE on the gels with the different
solidification times.
gel solidification time
number of matched
peptides
(hours)
1
3
7
7
6
8
12
24
8
13
The repeating unit 72 m/z belongs to acrylamide
monomer residue [MH]+, 44 m/z belongs to the
Figure 3. The comparison of prohibition PMF spectrum from the two gels with different solidification time (1 h and 24 h).
1076

I. Talian et al.
Figure 4. Detail of prohibition PMF spectrum with the repeating acrylamide monomer units and its fragments. Underlined masses are peptides
of prohibition origin.
observed. In some cases the peptide signal even
disappeared especially for the heavier peptides which
are usually used in MS/MS identification. As an example
the protein prohibition isolated from the MCF 7 cell
line was chosen. The number of matched peptides for
prohibition rises from 5 (in the one hour solidification
time gel) to 10 (in the gel solidification time of 24 hours)
and for protein cytochrome c the number of matched
peptides rises from 7 to 13. The prohibition sequence
coverage was improved from 16% to 41.5% and the
intensity of the MS spectrum was also considerably
improved. The possible explanation of this behavior is
in the protein/peptide – surface/acrylamide interaction
during the desorption/ionization in MALDI analysis. The
acrylamide polymerization during the laser pulse was
also observed and can be seen on the prohibition MS
Figure 5. Acrylamide fragmentation scheme.
spectrum where repeating acrylamide monomer units
[O = C - NH2]+ group and 28 m/z to this group [C = O]+. are marked. The problems with the un-polymerized
The scheme of acrylamide fragmentation is shown in acrylamide monomers can be partially overcome by
the Fig. 5.
utilizing pipette tips with incorporated packing material
In the case where the un-polymerized acrylamide such as C18. However, some strongly hydrophobic or
reacts with the protein already during the PAGE the hydrophilic peptides can be lost.
mass spectrum usually contains few peptide masses
and the others do not correspond to the peptide masses
of this certain protein (spectrum not shown). These
peptide masses are shifted to higher masses due to
Acknowledgement
acrylamide reaction with cysteine (carbamidoethylation
of cysteine).
The authors would like to acknowledge The Scientific
Grant Agency at the Ministry of Education, Slovak
Republic (VEGA 1/0802/09) and The Agency of the
Slovak Ministry of Education for the Structural Funds
of the EU, under projects ITMS: 26220220143, ITMS:
26220120039 and ITMS: 26220120067for the financial
support of the research. The authors would also like to
acknowledge Dr. Marianna Trebuňová for her help with
cell line cultivation and Dr. Galina Laputková for her help
with gel electrophoresis experiments.
4. Conclusions
The presented results clearly show the influence of
acrylamide monomer residues from 2D PAGE on the
peptide mass fingerprinting of separated proteins.
Considerable signal suppression of peptides was
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Impact of un-polymerized acrylamide monomer residues
onto protein identification by MALDI TOF MS
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