Download Protein Detection Methods in Proteomics Research

Bioscience Reports, Vol. 25, Nos. 1/2, February/April 2005 (Ó 2005)
DOI: 10.1007/s10540-005-2845-1
Protein Detection Methods in Proteomics Research
Reiner Westermeier1,3 and Rita Marouga2
In proteomics research chemical as well as physical methods are used to detect proteins
subsequently to their separation. Physical methods are mostly applied after chromatography. They are either based on spectroscopy like light absorption at certain wavelengths or
mass determination of peptides and their fragments with mass spectrometry. Chemical
methods are used after two-dimensional electrophoresis and employ staining with organic
dyes, metal chelates, fluorescent dyes, complexing with silver, or pre-labeling with fluorophores. In some cases autoradiography is still used. Since all of these techniques are very
different in terms of sensitivity, their usefulness for quantitative determinations varies
significantly. This review will describe the various protein detection methods applied to
electrophoresis gels.
KEY WORDS: Blotting; protein staining; protein labeling; two-dimensional electrophoresis; quantification; DIGE.
ABBREVIATONS: 2-D: two-dimensional; CCD: charge coupled device; DIGE: difference
gel electrophoresis; EDTA: Ethylenediaminetetraacetic acid; ICAT: isotope coded affinity
tag; MALDI ToF: matrix assisted laser desorption ionization time of flight; NMR: nuclear
magnetic resonance; PTM: post-translational modification; PAS: periodic acid/Schiff’s
reagent; PVDF: Polyvinylidenefluoride; SDS: sodium dodecyl sulphate; Tris: Tris(hydroxymethyl)-aminoethane.
INTRODUCTION
The selection of the appropriate protein detection method is highly important,
because the proteomics approach measures quantitative changes in expression levels
in biological samples. A number of very different techniques exist. Ideally the
detection limit should be as low as possible with an optimal signal to noise ratio. For
proper quantification of proteins in typical proteomics samples the detection method
should have a wide dynamic range and a wide linear relationship between the
quantity of protein and the staining intensity. The procedure should be easy and fast
to perform, non-toxic, environment-friendly, mass spectrometry-compatible, and
not too expensive. At present no protein detection technique meets all these
demands. It is therefore necessary to select the method which is optimal according to
the biological question, sample type and other methodical requirements.
1
GE-Healthcare BioSciences, Munzinger Strasse 9, D-79111, Freiburg, Germany.
GE-Healthcare BioSciences, Björkgatan 30 S-751 82, Uppsala, Sweden.
3
To whom correspondence should be addressed. E-mail: [email protected]
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2
0144-8463/05/0400-0019/0 Ó 2005 Springer Science+Business Media, Inc.
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Westermeier and Marouga
PROTEIN STAINING IN POLYACRYLAMIDE GELS
After two-dimensional (2-D) electrophoresis the proteins are detected in a SDS
polyacrylamide gel with small pore sizes, and the proteins are unfolded. Under this
condition it is easy to prevent the proteins from diffusion within and out of the matrix.
Fixing with highly concentrated trichloroacetic acid is therefore not necessary.
Coomassie Brilliant Blue Staining
Since its introduction by Fazekas de St. Groth et al. (1963) staining with the
organic dye Coomassie Brilliant Blue is still the most frequently employed method
for protein detection in polyacrylamide electrophoresis gels. This anionic triphenylmethane dye is used in two modifications: Coomassie R-250 (Red tint) and
Coomassie G-250 (Green tint), which has two additional methyl groups. In presence
of an acidic medium these dyes stick to the amino groups of the proteins by
electrostatic and hydrophobic interactions. In the original protocols the gels were
placed for a certain time into a staining solution of 0.1% Coomassie dye dissolved in
45% (v/v) methanol, 45% (v/v) water and 10% acetic acid; the background was
destained with 25% (v/v) methanol, 65% (v/v) water and 10% acetic acid. Sometimes methanol was replaced by ethanol or isopropanol. Practice has shown that this
procedure is not reproducible and is not very reliable for quantification. Because of
the presence of alcohol some of the proteins release the dye during the background
destaining process. Some proteins, like for instance collagen, destain faster than the
polyacrylamide gel.
Colloidal Coomassie Blue staining shows much better reproducibility and can
reach very high sensitivity, when it is applied long enough or repeatedly. There is no
background destaining. Because it is an endpoint method, highly reproducible results
are obtained. The most established procedure is the improved protocol by Neuhoff
et al. (1988). First the gel is placed in fixing solution (1.3% (w/v) o-phosphoric acid
and 20% methanol (v/v) in water) for 1 hr. The staining solution is prepared by
slowly adding 100 g of ammonium sulfate to 980 ml of a 2% H3PO4 (w/v) solution
until it has completely dissolved. Then a solution of 1 g Coomassie Brilliant Blue
G-250 solution in 20 ml of water is added. This staining solution must not be filtered,
but shaken before use. Staining is carried out overnight on an orbital shaker with
160 ml of staining solution plus 40 ml of methanol, which is added during staining.
Then it is placed into a neutralization solution, composed of 0.1 mol/l Tris/H3PO4
buffer, pH 6.5, for 3 min. It is rinsed for a maximum of 1 min in 25% (v/v) aqueous
methanol. The protein–dye complex is stabilized in 20% aqueous ammonium sulfate
for one day and then staining and the other steps are repeated for two more times.
The gel can remain in staining or stabilizing solution for several days without any
effect on the results. For optimal sensitivity with a detection limit of several
nanogram per spot the total procedure takes about 1 week.
The modification according to Anderson et al. (1991) is less labor-intensive, but
should give comparable results regarding sensitivity. First the gel is fixed for at least
3 hr in 50% (v/v) ethanol/3% (w/v) phosphoric acid and then washed three times for
29 min in water. After pre-incubation for 1 hr in 34% (v/v) methanol/3% (w/v)
phosphoric acid/17% (w/v) ammonium sulfate solution, 0.53 g Coomassie Brilliant
Protein Detection Methods
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Blue G-250 powder is added per 1.5 l solution. The gel is stained for 4–5 days on an
orbital shaker whereby the end-point is reached after 4–5 days. Subsequently, the gel
is washed a few times in water to remove background staining. On 1.5 mm gels a
detection limit of about 15 ng per spot is reached.
Another useful procedure is fast hot Coomassie staining with 0.025% (w/v)
Coomassie R-350 dissolved in 10% (v/v) acetic acid. Either it is performed in a metal
tray with a lid heated to 50°C for 15 min or the staining solution is heated to 90°C,
poured over the gel in a stainless steel tray with a lid. This tray covered with a lid is
placed on a laboratory shaker for 10 min. Destaining is performed in a tray on a
rocking table in 10% acetic acid for at least 2 hr at room temperature. Either the
solution is changed several times, recycled by pouring it through a filter filled with
activated charcoal, or a paper towel is placed into the destaining solution to absorb
the Coomassie dye. Staining and destaining solutions can be used repeatedly;
however, the sensitivity is not as high as with the two colloidal procedures.
The advantages of Coomassie staining methods are the quantitative binding of
the dye to proteins, the low price and the good reproducibility. For mass
spectrometry analysis the dye is removed from the gel plug with bicarbonate prior to
tryptic digestion. When a protein is detectable with Coomassie Brilliant Blue, as a
rule of thump, enough protein is present for appropriate mass spectrometry analysis
with MALDI ToF. The disadvantages are the long staining times, the relatively low
sensitivity, and the narrow dynamic range.
Copper Staining
Although staining with 0.3 M copper chloride solution according to Lee et al.
(1987) is faster, easier, and more sensitive than Coomassie staining, it has never
become well accepted for applications in proteomics research.
Negative Imidazol SDS Zinc Staining
For the matter of further analysis of the detected proteins it is appealing to apply
a detection method, which does not modify the protein but visualizes the background.
A complex salt of zinc, SDS and imidazole precipitates in the polyacrylamide gel and
forms an opaque white background, but stays soluble in presence of a protein spot
(Fernandez-Patron et al., 1992). The protein zones become visible against a dark
background. The sensitivity of detection (which is at a few nanograms) lies between
Coomassie Blue and silver staining. The peptide yield after in-gel digestion is said to
be higher then after direct staining of proteins. The salt precipitate can be dissolved
with a Tris–EDTA buffer, which allows electrophoretic transfer of the proteins for
Western blotting. Although it is claimed that the procedure allows quantitative
determination of proteins (Ferreras et al., 1993), this statement is disputed because
only the background is detected directly, but not the protein.
Silver Staining
Detecting proteins with silver staining is widely used, because the sensitivity is
below 1 ng per spot, the costs for reagents are relatively low, and—although it is a
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multistep procedure—the results are available relatively quickly. Ideally the spots are
dark brown to black on a light beige background. Since the introduction of silver
staining in polyacrylamide gels by Merrill et al. (1979) about one hundred different
modifications of silver staining protocols have been published. An extensive review
of silver staining methods has been written by Rabilloud et al. (1994). In principle,
the protocols can be divided into three groups:
Colloidal fast staining in presence of tungstosilicic acid based on the first silver
staining protocol in agarose gels according to Kerenyi and Gallyas (1972). These
protocols have the lowest sensitivity of the three groups, but are compatible with
mass spectrometry.
The principle of a silver nitrate staining protocol works as follows: First the
proteins are fixed; SDS and buffer components are washed out with 40% ethanol
and 10% acetic acid. The next step combines cross linking of the proteins in the
matrix with glutardialdehyde and sensitizing with sodium thiosulfate in presence of
sodium acetate. After a number of washes with distilled water with exact timing, the
gel is placed into a solution containing 0.25% silver nitrate and a low amount of
formaldehyde. Development is performed with formaldehyde at basic pH value,
which is achieved with sodium carbonate. The reaction is stopped by placing the gel
either into 5% acetic acid or 1.5% EDTA. In practice the following two protocols
(according to Heukeshoven and Dernick, 1985; Blum et al., 1987) work best and
show the highest sensitivities (down to 100 pg per spot). When spots need to be
analyzed further with mass spectrometry, the glutardialdehyde must be omitted from
the sensitizer solution and the formaldehyde from the silver nitrate solution
(Shevchenko et al., 1996). In order to obtain efficient staining, the development step
Fig. 1. 2-D electrophoresis gel stained with the silver nitrate method
according to Heukeshoven and Dernick. Protein extract from Brassica oleracea (cauliflower) seeds separated in an immobilized pH
gradient 4–7 over a distance of 24 cm followed by SDS polyacrylamide gel electrophoresis with 18 cm separation distance.
Protein Detection Methods
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is then performed with the double amount of formaldehyde compared to standard
protocols. This modification reduces the sensitivity to about one fourth. Figure 1
shows a 2-D electrophoresis gel of a protein extract from cauliflower seeds stained
with silver nitrate according to Heukeshoven and Dernick (1985).
Ammoniacal silver complex protocols look in principle like this: Fixing is often
combined with the sensitizer, which in this case is naphthalene disulfonate. After
several washing steps the gel is placed into ammoniacal silver solution. The development is performed under acidic conditions in citric acid with formaldehyde. The
reaction is stopped with acetic acid and ethanolamine. This procedure was
introduced by Eschenbruch and Bürk (1982) and further improved by many different
groups, mainly by Rabilloud (1992). This method is critical for forming silver
precipitation on the gel surface (called ‘‘mirrors’’) caused by traces of impurities in
one of the solutions, and needs a much higher amount of silver nitrate than the silver
nitrate based protocols.
In general, silver staining detects the proteins mainly on the gel surface. This is
also the reason why further analysis of silver stained proteins is still possible. Silver
staining is a multistep procedure. Because it is not an endpoint procedure, the
staining intensities can vary from gel to gel. The use of a staining automat makes the
procedure much more convenient and increases its reproducibility substantially.
Unfortunately silver staining shows only a narrow dynamic range and is not reliable
for quantification. An often observed phenomenon is that highly abundant proteins
produce spots with a yellow centre. As shown in Fig. 2, these spots look like volcano
craters or donuts, and create problems for image analysis software for qualitative
and quantitative analysis. This effect can be repaired when the gel is submitted to
blue toning after silver staining (Berson, 1983). This procedure further increases the
sensitivity of silver staining.
Fig. 2. Effect of high protein concentration in some spots on silver staining. (a) 2-D view
of a gel section. (b) 3-D view of the same section. The centres of these spots are yellow and
lighter than the edges.
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Fluorescence Staining
Fluorescence dyes show very wide linear dynamic ranges, over four orders of
magnitude. The results are highly reproducible, because fluorescent staining is an
endpoint method. Since the introduction of Nile Red staining (Bermudez et al.,
1994) various dyes have been developed during the last few years with markedly
different sensitivities and properties such as the family of SyproÒ dyes (Orange, Red,
Tangerine and Ruby) (Steinberg et al., 1996; Berggren et al., 2002). Whereas the
sensitivities of SyproÒ Orange, Red, and Tangerine are similar to Coomassie Blue,
SyproÒ Ruby can detect down to about 1 ng of protein in a spot. Rabilloud et al.
(2001) published a protocol for a ruthenium complex fluorescence staining with high
sensitivity. A new dye, called Deep PurpleÔ, contains the fluorophore ‘‘epicocconone’’ from the fungus Epicoccum nigrus (Mackintosh et al., 2003) and is even more
sensitive than SyproÒ Ruby, down to a few hundred picograms. Besides the high
sensitivity, the latter dye has some more advantages: it does not create speckles in the
background and is a natural product, which is easy to dispose of.
Fluorescence staining is the optimum method for combining high sensitivity of
detection with reliable quantification over a wide linear dynamic range. However, the
dyes are more expensive than those used for visual staining; a fluorescence scanner is
needed to visualize the spots and to acquire the image for image analysis (see section
image acquisition below). Figure 3 shows the comparison of silver stained and
fluorescence-labeled 2-D electrophoresis gels. At the first glance the fluorescence gel
seems to show less resolution than the silver stained gel. However, if the peaks are
displayed in a 3-D view as shown in Fig. 4, the wide dynamic range of the detection
method becomes obvious.
Specific Stains for Post-translational Modifications
For studies of the two most important post-translational modifications, glycosylation and phosphorylation, specific stains can be used. The periodic acid Schiff’s
Fig. 3. Comparison of an image of a fluorescent (left hand side) and silver stained gel
(right hand side). The silver staining gel image makes believe, that it has a higher resolution than fluorescent staining. Only the 3-D view (see Fig. 4) will reveal the high quality
result of the fluorescence staining, which is due to its wide dynamic range.
Protein Detection Methods
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Fig. 4. Two- (insert) and 3-D view of a few protein spot detected with fluorescent
labeling.
reagent procedure for specific detection of glycoproteins, introduced by Zacharius
et al. (1969) has become much more sensitive by coupling the reaction with
fluorescence (Eckhardt et al., 1976). Recently, a commercial product called
‘‘Pro-QÒ-Emerald’’ (Hart et al., 2003) became available and a further fluorescence
dye called ‘‘Pro-QÒ-Diamond’’ was introduced for specific detection of phosphorylated proteins (Steinberg et al., 2003). It is possible to stain a gel sequentially with
different specific dyes and finally with a total stain.
PRE-LABELING OF PROTEINS WITH FLUOROPHORES
In 2-D electrophoresis, proteins can be pre-labeled with a fluorescent dye after
the isoelectric focusing step prior to SDS polyacrylamide gel electrophoresis (Urwin
and Jackson, 1991) or prior to isoelectric focusing with monobromobimane (Urwin
and Jackson, 1993). The spots can be directly scanned in the gel (while it is still in the
cassette) when low-fluorescent glass is used. Like other fluorescent dyes, also this
label exhibits a high sensitivity and a wide dynamic range of the signal.
A quantum leap in 2-D gel electrophoresis methodology has been achieved
by the introduction of size and charge matched cyanine dyes (CyDyeÔ DIGE
Fluors) with different excitation and emission wavelengths as protein labels for
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different samples (Ünlü et al., 1997). The method, called ‘‘difference gel electrophoresis’’ (DIGE), allows multiplexing of samples and the use of an internal
standard, which is created by using one of the labels for a pooled mixture of all
samples. This method leads to highly accurate qualitative and quantitative results,
because gel-to-gel variations are eliminated. Figure 5 shows the workflow of
difference gel electrophoresis. Two alternative concepts exist: minimum labeling
by attaching the dye to the free amino group of lysine residues and saturation
labeling of all cysteine residues. In the first variant only 3–5% of the total
proteins will receive a label, which insures that only singly labeled proteins will be
detected. In this case the labeled proteins co-migrate with the non-labeled proteins in both separation directions and the resulting 2-D image looks like the one
achieved with post-staining. Typically, 400 qmol/l of one of the three available
cyanine dyes is added to 50 lg protein of each sample and the pooled standard.
The sensitivity of detection is comparable to a sensitive silver staining method.
With the second variant a considerable increase in sensitivity is obtained. 2-D
patterns resulting from down to 1 lg total protein load can be visualized. Because
all available cysteine residues are labeled, many multiply labeled proteins exist.
The resulting spot patterns are different from those achieved with post-stained or
lysine labeled proteins. After scanning the gel at different wavelengths, the comigrated protein spots of different sample origins are co-detected using dedicated
software, which allows very fast and completely automatic pattern evaluation thus
avoiding any bias introduced by an operator. With the help of the pattern created
by the internal standard, which is run in each gel, the patterns of different gels
can easily be matched and the sample spot volumes can be normalized to the spot
volume of the standard. This results in very accurate protein difference ratios with
high statistical confidence (Alban et al., 2003; Friedman et al., 2004).
Fig. 5. Schematic representation of 2-D difference gel electrophoresis in one gel. Example for two
samples. When more samples are to be compared, more gels are run, every time including the
internal standard, which is a mixture of pooled aliquots of all samples.
Protein Detection Methods
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ISOTOPE LABELING
Radioactive Labeling
In the pioneering paper by ÓFarrell (1975) on high-resolution 2-D electrophoresis it was shown that autoradiography is 100–1000 times more sensitive than
staining with Coomassie Brilliant Blue. The labeling is carried out predominantly in
living cells, in bacteria, yeast or tissue cultures with the help of 35S-methionine and/
or 32P orthophosphate. It is also possible to apply specific labeling, for instance with
32
PO3)
for the detection of phosphoproteins. Post-synthetical radiolabeling, for
4
instance by iodination with 125I or alkylation of cysteine residues with 14C- or
3
H-iodoacetamide is problematic due to problems with reproducibility and sensitivity. Because the wet gel layer quenches the radioactivity signal, the gels need to be
dried prior to exposure to an X-ray film or a storage phosphor screen.
Fluorography
Wet gels can be used and the sensitivity can be increased with scintillation
autoradiography, also called fluorography. The gel with 35S, 14C- or 3H labeled
proteins is impregnated after fixation with a fluor like 2,5-diphenyloxacole. The
low-energy b-particles excite the fluor molecules, which will then emit a light signal.
Even though very high sensitivity of detection seems to be desirable, these
methods are not very useful for proteomics applications because the spots of low
expression do not contain enough protein for subsequent mass spectrometry
analysis. Furthermore, because of environmental reasons, autoradiography and
fluorography are becoming less important.
Stable Isotope Labeling
Stable isotope labeling methods for the analysis of quantitative differences
between two samples can only be used when a high-resolution mass spectrometer is
available. For such a differential approach, cells can be labeled by adapting
techniques developed for NMR by using ammonium salt as nitrogen source with 14N
vs. 15N or glucose as carbon source for 12C vs. 13C labeling. The detection is carried
out by mass spectrometry of the tryptic digests of protein spots. Because ionization is
equal for the same peptides containing the different isotopes, the heights of the
shifted peaks can be used to measure quantitative ratios (Oda et al., 1999). For
stable isotope tagging of proteins after synthesis, isotope coded affinity tag (ICAT)
labeling, known from differential mass spectrometry analysis, has also successfully
been applied for 2-D electrophoresis (Smolka et al., 2002).
BLOTTING TECHNIQUES
Alternatively to gel-based methods, proteins can be detected on immobilizing
membranes, to which they have been transferred after electrophoretic separation.
This transfer is usually carried out with electrophoretic force, because diffusion or
capillary forces are not sufficiently efficient to mobilize the proteins from inside a
polyacrylamide gel (Towbin et al., 1979). For electro blotting either the blotting
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Westermeier and Marouga
gel-membrane sandwich is clamped between grids and inserted into a buffer tank
with platinum electrode wires at the walls (‘‘tank blotting’’) or the blotting sandwich
is placed between two horizontal plate electrodes made from graphite or platinumcoated steel meshes (‘‘semi-dry blotting’’). The membrane material used is mostly
nitrocellulose or PVDF (Polyvinylidenefluoride). Both membrane types are available
with different pore sizes and mechanical strengths, and they have different binding
properties. Two technical transfer problems can limit the performance of blotting
methods: Some proteins do not transfer efficiently, particularly large ones
(>80,000 Da) and other proteins are not bound efficiently by the membrane and
migrate through, mostly small ones. There are several possibilities of protein
detection on blotting membranes:
Total Protein Staining
General protein staining of blotted proteins is mainly applied for locating and
confirming the spot position and identity in connection with special protein probing
or further protein analysis. Usually it is not employed as an alternative to staining of
proteins in the gel, because it is too labor-intensive and bears too many risks of
inefficient transfer. If a high sensitivity is not required, the reversible Ponceau red
staining according to Salinovich and Montelaro (1986) is the standard procedure,
showing a detection limit in the range of 1 lg/spot. Amido Black, Coomassie
Brilliant Blue, Fast Green FCF, and Indian ink are also used for staining blotting
membranes. The most sensitive techniques are colloidal gold (Moeremans et al.,
1985) and Deep PurpleÔ staining with detection limits down to 3 ng/spot. SyproÒ
Rose Plus and SyproÒ Ruby fluorescent dyes show sensitivities of about 12 ng/spot.
It should be noted that during the blotting transfer from the separation gel to the
membrane some proteins can get lost, either due to incomplete transfer or
non-quantitative binding properties of the membrane. The protein detection limits
given above are measured from the amount of protein loaded to the gel; they do not
mean the actual amount on the blotting membrane.
Special Protein Staining
The specific gel stains for phosphoprotein and glycoprotein detection can also
be applied on blotting membranes. The steps are much shorter than in gels, because
the proteins are easily accessible on the surface of the membrane. Specific glycan
detection kits for blotting membranes are offered by Roche Applied Sciences:
glycoproteins are probed with digoxigenin, which is then probed with alkaline
phosphatase labeled antidigoxigenin. The visualization is performed by colorimetric
detection of the alkaline phosphatase with Nitroblue tetrazolium. According to the
brochure by Roche, the sensitivity of detection lies in the range of several nanograms
per protein band, dependent on the individual protein analyzed. This technique can
be combined with selected lectins to probe for specific glycoconjugate structures.
Immuno Blotting
When electrophoretic protein blotting is followed by immunodetection, it is
frequently called ‘‘Western Blotting’’. After the transfer the free binding sites on the
Protein Detection Methods
29
membrane must be blocked with a mixture of macromolecular substances, which do
not participate in the visualization reaction. Often skim milk powder, casein
hydrolysate, or fish gelatin is used. The probing for individual proteins is performed
with immunoglobulins or monoclonal antibodies. Up to four times reprobing for
different antigens is possible. For visualization a secondary antibody is used, which
is conjugated either to gold, horseradish peroxidase, alkaline phosphatase or an
avidin--biotin-enzyme complex for amplification of the signal. The conjugated
enzymes are detected with the respective functional zymogram staining, which has a
high sensitivity. The highest sensitivity without using radioactivity is obtained with
enhanced chemiluminescence detection methods (Laing, 1986). The signal is detected
by exposing the membrane to an X-ray film or placing it into a video documentation
system for a certain time period, which is in principle a CCD camera in a dark
cabinet. With a further developed chemiluminescence method (ECL AdvanceÔ)
down to 0.1 femtogram of antigen can be detected. The blotting membranes can also
be probed with a non-antibody protein to study specific protein–protein interactions
(Burgess et al., 2000). The probing protein is then detected with a labeled antibody.
This variant is either called ‘‘Far-Western blotting’’ or protein overlay detection.
Protein Identification
Blotting on PVDF or modified glass fiber membranes has been widely used for
protein identification by amino acid composition analysis, N-terminal amino acid
sequencing, peptide mass fingerprinting and sequencing with mass spectrometry.
With the extended possibilities offered by new mass spectrometry technologies the
blotting step can be omitted; proteins are digested inside the gel layer and the eluted
peptides are submitted to mass spectrometry analysis.
IMAGE ACQUISITION
The complex protein patterns obtained in proteomics research require computeraided image analysis. Therefore the image needs to be converted into digital format.
Camera Systems
For chemiluminescence detection usually a camera in a dark cabinet is
employed. When all proteins need to be detected, only scanning cameras can provide
enough resolution for an appropriate image analysis. However, the acquired
adjacent image patches need to be stitched together electronically, which can be a
source of errors in the qualitative and quantitative evaluation.
Scanners (Densitometers)
Point and line scanners are much better suited for the acquisition of
high-resolution protein patterns. Gels with visible protein zones need to be scanned
with light transmission in order to obtain quantitative reliable signals. This is possible with high-end, liquid-insulated desktop line scanners. Before scanning the
instrument must be calibrated. For appropriate image analysis is important to scan
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Westermeier and Marouga
the gels in grayscale mode with at least 16 bit signal depth. For fluorescence staining
or labeling special fluorescence scanners are needed. Multiple fluorescence scanners
are usually point scanners with laser light sources with different wavelengths. The
best instruments use confocal optics in order to exclude stray light signals and to get
reliable quantitative signals with high resolution. It should also be noted that the
wide dynamic range, obtained with a detection method, can only be maintained
when the scanner offers a wide dynamic range as well. Multiple fluorescence scanners
can also be used to measure radioactivity with the usage of storage phosphor screens.
IMAGE ANALYSIS
Software for image analysis should evaluate protein patterns reliably and
reproducibly. Human interference must be kept to a minimum, because of two major
reasons: image analysis with manual adjustments can be a severe bottleneck in the
workflow and it is an additional source of variation. The main steps are spot
detection, normalization and spot matching between different gels, and finally
quantitative comparisons of spot volumes. The significance of changes in protein
expression levels detected in different gels, and the confidence level of a measured
quantitative ratio are checked with statistic tools, which are usually supplied with
professional image analysis software. Before results are published, the protein spots
of interest must be further analyzed. It can always happen that an electrophoresis
band or spot contains more than one protein due to insufficient resolution. In such
cases the measured spot volume ratios will not reflect the real situation. Usually this
analysis is performed with mass spectrometry.
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