1. science
2. chemistry

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DOI 10.1002/pmic.200600184
Proteomics 2007, 7, 2340–2349
RESEARCH ARTICLE
An improved method of sample preparation on
AnchorChip™ targets for MALDI-MS and MS/MS and its
application in the liver proteome project
Xumin Zhang1, 2, Liang Shi1, Shaokung Shu1, Yuan Wang1, Kang Zhao1, Ningzhi Xu1,
Siqi Liu1, 3 and Peter Roepstorff2*
1
Beijing Genomics institute, Chinese Academy of Science, Beijing, China
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
3
Departement of Medicine, University of Louisville, Louisville, KY, USA
2
An improved method for sample preparation for MALDI-MS and MS/MS using AnchorChip™
targets is presented. The method, termed the SMW method (sample, matrix wash), results in
better sensitivity for peptide mass fingerprinting as well as for sequencing by MS/MS than previously published methods. The method allows up-concentration and desalting directly on the
mass spectrometric target and should be amenable for automation. A draw back caused by
extensive oxidation of methionine and tryptophan in the SMW method can be alleviated by the
addition of n-octyl glucopyranoside and DTT to the sample solution. The method was validated
for protein identification from a 2-DE based liver proteome study. The SMW method resulted in
identification of many more proteins and in most cases with a better score than the previously
published methods.
Received: March 13, 2006
Revised: February 9, 2007
Accepted: April 9, 2007
Keywords:
2-DE / AnchorChip target / Liver proteome / MALDI-TOF-MS / Sample preparation
1. Introduction
Correspondence: Dr. Siqi Liu, Beijing Genomics Institute, Chinese Academy of Sciences, Beijing Airport Industrial Zone B-6,
Beijing 101300, China
E-mail: siqiliu@genomics.org.cn
Fax: 186-10-8049-8676
using one single step of sample preparation. They are now
widely used for protein identification in proteomics.
Over the years numerous methods of sample preparation
for MALDI have been described. These include the drieddroplet (DD) [1], crushed-crystal [5], thin-layer (TL) [6], and
sandwich [7] methods. The latter two methods include ontarget washing to remove salts. On target washing in combination with the DD method has also been reported [8]. In
spite of the fact that MALDI is rather tolerant to impurities, it
will often be advantageous to up-concentrate and desalt peptide samples prior to analysis. This is frequently achieved
using miniaturized columns packed with RP resin, e.g.
microcolumns [9] and ZipTip tips [10]. The performance of
the different sample preparation methods has been described by Kussmann et al. [11]. Unfortunately, none of these
methods are convenient for large-scale proteome analysis
because they are time consuming and laborious.
Abbreviations: DD, dried droplet; n-OGP, N-octyl glucopyranoside; SMW, sample matrix wash; TL, thin-layer
* Additional corresponding author: Dr Peter Roepstorff
E-mail: roe@bmb.sdu.dk
MALDI has gained widespread use in protein studies since
its introduction in the late 1980’s by Karas and Hillenkamp
[1, 2]. Due to high sensitivity, fast and easy sample preparation, high tolerance to impurities and easy data analysis, it is
now one of the key analytical techniques in proteomics. The
recent introduction of MALDI-TOF/TOF tandem mass spectrometers [3, 4] has greatly increased the utility of MALDIMS in proteome analysis because these instruments allow
generation of PMF’s and peptide sequence information
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Technology
Proteomics 2007, 7, 2340–2349
Recently, prestructured sample supports so called
AnchorChip™ targets were introduced by Bruker Daltonics
[12]. Compared with the use of microcolumns these targets
offer sensitivity advantages because they allow sample upconcentration by reducing the size of the sample spot even
when applying rather large sample volumes. The original
protocol for sample preparation on AnchorChip™ targets was
based on the use of DHB as matrix. It included a rather time
consuming desalting step. Two alternative procedures using
CHCA as matrix and on-target desalting have been described
[13, 14]. These two methods resulted in some improvement
over the original protocol because a separate desalting step
prior to applying the sample could be eliminated. However,
the desalting efficiency and sample re-crystallization to generate a homogeneous sample deposit were not satisfactorily
solved. Since the Bruker Ultraflex TOF and TOF/TOF
instruments in combination with AnchorChip™ targets are
widely used in proteomics studies we decided to try to overcome these limitations.
Here, we present an improved method for sample preparation on AnchorChip™ targets, which is different from the
previously published sample preparation methods [13–15].
Our improved method is fast and efficient and offers
improved performance for protein identification using peptide mass fingerprinting as well as sequencing by MS/MS as
demonstrated with examples from the liver proteome project.
2
Materials and methods
2.1 Materials and reagents
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tocols for animal operation were approved by the CAS Committee for Animal Experimentation. The mice were anesthetized with pentobarbital sodium and sacrificed for removing
livers. Fresh liver tissues were placed in a clean mortar containing liquid nitrogen and finely ground. The resulting
powders were dissolved and sonicated in the lysis buffer
containing 10 mM Tris-HCl, pH 7.4, 8 M urea, 4% w/v
CHAPS, 10 mM DTT, 1 mM PMSF, and 2 mM EDTA, followed by centrifugation at 15 0006g for 30 min. The supernatants were stored at 2807C until use. The protein concentrations were determined with the Bradford assay.
2.3 2-DE
The protein solutions were mixed with rehydration buffer
containing 8 M urea, 2% w/v CHAPS, 20 mM DTT, 0.5%
IPGphor buffer (pH 3.0–10.0, NL), and 0.002% bromophenol blue, and 18 cm IPG strips were rehydrated overnight
with appropriate amounts of protein solution (100 ug protein/gel). IEF was carried out at 56 kVh at 207C using an
IPGphor (Amersham, Uppsala, Sweden). The focused strips
were equilibrated in the buffer with 6 M urea, 50 mM TrisHCl, 30% glycerol, 2% SDS, and a trace of bromophenol
blue, and were subsequently treated by reduction with DTT
and alkylation with iodoacetamide. The treated strips were
transferred onto 12% uniform SDS-polyacrylamide gels
using the Ettan DALT II system (Amersham) with a programmable power supply. Gels were run with 2.5 W per gel
for 30 min followed by 15 W per gel until the bromophenol
blue dye reached the bottom of the gel. The separated proteins were visualized by silver nitrate staining according to
the protocol from Amersham with an additional developing
step to reduce the background staining.
CHCA was obtained from Aldrich (Steinheim, Germany);
Pepmix containing Angiotensin II, (DRVYIHPF,
[M1H]1 = 1046.5420), Angiotensin I (DRVYIHPFHL,
[M1H]1 = 1296.6853), Substance P (RPKPQQFFGLM-NH2,
[M1H]1 = 1347.7361), Bombesin (pEQRLGNQWAVGHLM-NH2, [M1H]1 = 1619.8230), ACTH clip 1–17 (SYSMEHFRWGKPVGKKR, [M1H]1 = 2093.0868), ACTH
clip 18–39 (RPVKVYPNGAEDESAEAFPLEF, [M1H]1 =
2465.1990), Somatostatin 28 (SANSNPAMAPRERKAGCKNFWKTFTSC, [M1H]1 = 3147.4714) was from Bruker
Daltonics (Part No. 20 61 95, Bremen, Germany). Modified
porcine trypsin (sequencing grade) was from Promega
(Madison, WI, USA). All of solvents used were sequence
grade from Sigma (Deisenhofen, Germany). Water was purified on a Milli-Q system (Millipore, Bedford, MA, USA).
The tubes of 0.5 mL were Safe-Lock™ tubes from Eppendorf
(Hamburg, Germany), and 1.5 mL tubes were from Biozym
Diagnostic (Hessisch Oldendorf, Germany).
Spots were excised manually from silver-stained 2-DE gels
and subjected to in-gel trypsin digestion. Briefly, the spots
were successively washed with water and 50% ACN. The
proteins were submitted to an additional reduction and
alkylation step using 10 mM DTT at 567C for 1 h followed by
55 mM iodoacetamide in the dark at room temperature for
45 min. Finally, the gel pieces were thoroughly washed with
25 mM NH4HCO3 in H2O/ACN (50/50 v/v) and subsequently dried in a Speedvac. The dried gel spots were reswelled by addition of a small volume of digestion buffer
(25 mM NH4HCO3 and 10 ng/mL of trypsin). After 30 min of
incubation on an ice bath, 20 mL of 25 mM NH4HCO3 were
added to cover the gel piece. Digestion was performed over
night at 377C and terminated by addition of 2 mL of 5% TFA.
2.2 Extraction of mouse liver proteins
2.5 Sample preparation for MALDI analysis
Male mice of the C57BL/6J strain (7–9 weeks of age) were
purchased from Beijing Laboratory of Animal Center and
were held under specific pathogen-free conditions. The pro-
A stock solution of matrix was prepared by dissolving CHCA
(10 mg/mL) in 70% ACN and 0.1% TFA. In the present study
600 mm spot size AnchorChip™ targets were used. For these
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.4 In-gel digestion
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X. Zhang et al.
targets, the matrix stock solution was diluted to 0.5 mg/mL
with freshly prepared solution containing 90% ACN and
0.1% TFA just prior to sample preparation. To ensure the
quality of the matrix solution, 0.8 mL was applied on the
target prior to applying a series of samples and the formed
matrix deposit examined for crystal homogeneity under a
microscope. A homogeneous deposit of small matrix crystals in the hydrophilic center should be observed similar to
the one illustrated in Fig. 1A. If this is not the case new
matrix solution must be prepared. One microliter of the
supernatant from the in-gel digestions was loaded on the
target and allowed to dry completely (the drying time is
about 10–20 min, depending on the room temperature and
humidity). Then 0.8 mL of matrix solution was applied to the
same spot and left to dry for 2 min. Subsequently, a 1 mL
drop of 0.5% TFA was applied on the matrix deposit. After
30 s, the remaining solution was removed with a pipette.
This washing procedure can be repeated if needed. In a
high throughput analysis, the target plate was simply
washed by dipping it into a 0.02% TFA for 20 s followed by
drying.
The two previously published methods for loading peptide digests onto AnchorChip™ targets, the TL affinity preparation method according to Gobom et al. and the DD preparation according to Thomas et al were performed as described in the AnchorChip™ manual respectively in ref. 13
[12, 13].
2.6 MS analysis
Mass spectra and tandem mass spectra were obtained on a
Ultraflex TOF/TOF mass spectrometer (Bruker). Positively
charged ions were analyzed in the reflector mode, using
delayed extraction. Typically 100 shots were accumulated per
spectrum in MS mode and 400 shots in MS/MS mode. The
spectra were processed using the FlexAnalysis 2.2 and BioTools 2.2 software tools (Bruker).
Proteomics 2007, 7, 2340–2349
2.7 Database search
Protein identification was performed using the MASCOT
software (http://www.matrixscience.com) to search the
NCBInr database with mouse as taxonomy. All peaks with a
S/N above 15 were included in the search. The following parameters were used for database searches: Monoisotopic
mass accuracy ,100 ppm, missed cleavages 1, carbamidomethylation of cysteine as fixed modification, oxidation of
methionine, N-terminal pyroglutamylation (peptide) and Nterminal acetylation (protein) as variable modifications. In
MS/MS mode, the fragment ion mass accuracy was set to
,0.7 Da.
3
Results and discussion
3.1 Comparison of matrix crystal formation from
three methods
The three methods for sample loading investigated in this
study were termed the sample matrix wash (SMW), the TL
method [13] and the DD method [14]. As shown in Fig. 1, the
three methods resulted in deposits with different crystal sizes
and matrix layer appearance. With the SMW method, a deposit
of small matrix crystals is obtained homogeneously distributed in the hydrophilic center (Fig. 1A). Using the TL method,
the matrix forms a uniform matrix layer without visible crystal
formation (Fig. 1B) With the DD method, the matrix crystals
are larger with uneven distribution over the chip surface and
partly also outside the hydrophilic center (Fig. 1C). For high
throughput analysis using MALDI-MS, a uniform crystal distribution over the hydrophilic center is preferable because this
allows automated recording of the spectra. Thus the SMWand
TL seem to be favored over the DD method.
To obtain good results with the SMW method, two
observations were made. Firstly, the matrix solution should
be freshly prepared. Secondly, sample and matrix solutions
Figure 1. Peptide-matrix crystal
layer obtained with the different
sample loading methods on
600 mm AnchorChip™ targets. (A)
the SMW, (B) the TL, and (C) the
DD method.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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might not dry completely on the chip when common plastic
tubes were used. After a subsequent washing procedure drying of the sample was possible showing that some component from the plastic tubes, which prevented the drying, was
removed in the washing procedure. However, the fine crystal
structure was destroyed and spreading of deposit outside the
hydrophilic center was observed. The use of Eppendorf SafeLock™ tubes (as recommended in the AnchorChip™ manual)
overcame this problem. Their use was found especially
important for preparation of the matrix solution.
with 0.02% TFA. No difference in sensitivity was observed
between the two washing methods independently of the
peptide amount added (data not shown).
Repeated independent analyses showed that the results
obtained with all three methods were reproducible in terms
of sensitivity and S/N. Thus for the SMW methods, six repeated analyses at the 0.4 fmol/mL level resulted in the following S/N ratios and SDs: m/z 1046, S/N 15.5 6 3.4; m/z
1296, S/N 39.5 6 11.4; m/z 1363, S/N 16.4 6 3.1; and m/z
2465, S/N 5.5 6 2.5.
3.2 Comparison of the sensitivity and reproducibility
of the three methods
3.3 Comparison of sample loss between the SMW
and TL method
To test the detection sensitivity, a series of solutions of the
standard pepmix were prepared containing 0.1, 0.4, 1, 4, and
10 fmol/mL of each peptide. For the SMW and TL methods,
1 mL of peptide solution was applied to the AnchorChip™
target whereas 1.2 mL were used for the DD method according to the published protocol. The results are shown in Fig. 2.
All seven peptides were observed at 10 fmol/mL, with
acceptable signal to noise (i.e. S/N better than 3 see inset in
Fig. 2) when the samples were loaded using the SMW or the
DD methods whereas only six peptides were observed using
the TL method. At 1 fmol/mL no peptide signals could be
detected with the TL method. Six and five peptide signals
were still observed using the SMW and DD methods,
respectively. Further dilution to 0.1 fmol/mL, still allowed
observation of three peptide signals using the SMW method
whereas no peptide signals were detected using the TL and
DD methods. The SMW method thus results in considerable
improved sensitivity compared to the previously published
methods for sample preparation on AnchorChipTM targets. A
comparison was also made with the high throughput washing method where washing in the SMW method was
accomplished by dipping the complete target into a beaker
The low sensitivity of the TL method might be due to poor
incorporation of the analyte in the thin layer of matrix with
concomitant increased losses during the washing procedure.
To investigate this, experiments were carried out to compare
the peptide loss in the SMW and TL methods. For the SMW
method, 1 mL washing solution (0.5% TFA) was applied to
the target after application of sample and matrix. After about
30 s, the droplet was removed and transferred to the neighboring position on the target and treated according to the
SMW method. For the TL method, the sample solution was
placed on the thin layer of matrix for 3 min. The remaining
solution was then removed and transferred to the neighboring position on the target and treated according to the SMW
method. The results obtained by analysis of the transferred
solutions are shown in Fig. 3A and B. Comparison between
Figs. 2 and 3 clearly reveals that the peptide loss using the
SMW method is insignificant whereas a majority of the
sample is lost during the TL preparation due to poor incorporation in the matrix layer. To test the efficiency of the
washing procedure in the SMW method, 1 mL of a solution
containing 25 mM Tris-HCl was dried on the target after
applying the sample solution, subsequently matrix solution
Figure 2. Comparison of the sensitivity of the three methods. Panel (A) SMW, (B) TL, and (C) DD. The peptide concentration is from top to
bottom: 10 fmol/mL, 4 fmol/mL, 1 fmol/mL, 0.4 fmol/mL, and 0.1 fmol/mL. The insets show the detailed profile of the peak at m/z 2465.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Proteomics 2007, 7, 2340–2349
Figure 3. Comparison of the sample loss with the three methods. Panel (A) The washing solution from the SMW method; (B) the sample
solution after application using the TL method; (C) the washing solution from the DD method. The loaded pepmix concentration is from top
to bottom: 10 fmol, 4 fmol, 1 fmol, 0.4 fmol and 0.1 fmol.
was applied and dried. Prior to washing, a clear deposit of
needle shaped Tris crystals was visible on the matrix surface.
Upon washing all these crystals had disappeared and the
matrix surface was similar to the one shown in Fig. 1A. A
similar investigation of the sample loss in the DD method
revealed that sample losses here as with the SMW method
are negligible (Fig. 3C). The sensitivity difference between
the SMW and the DD methods is most likely due to the better
focusing of the sample/matrix deposit in the former method
as shown in Fig. 1.
3.4 Comparison of MS/MS performance of the three
methods
Considerably larger amounts of sample is consumed to
record spectra in the MALDI-MS/MS mode compared to the
MALDI-MS mode due to the use of higher laser fluence in
the MS/MS mode. This means that it is important to establish the depletion of analyte as function of number laser
shots at a given position. For each sample preparation
method spectra were recorded for series of 50 laser shots
without moving the laser position on the target. With the
SMW method, the absolute intensity of the MS spectra was
almost unchanged within the first several 50 shots (Fig. 4A).
Moreover, the acceptable spectra were still obtained for shot
number 701 to 750. Using the DD method, the peptide signals dramatically decreased after the first 50 shots (Fig. 4B)
and could hardly be observed after 300 shots. With the TL
method, the signal disappeared after the first 50 shots, (data
not shown). The latter observation is not surprising since
most of the sample will be close to or at the surface with the
TL method. It is not quite clear why the analyte was quickly
depleted with the DD method even after first searching for
good spots.
To compare the sensitivity in MS/MS mode of the three
sample preparation methods, pepmix (10, 4 and 1 fmol/mL)
were loaded with all three methods. Two peptide signals
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(m/z 2465.16 and m/z 1296.68) were chosen to evaluate the
MS/MS performance. As shown in Fig. 5, satisfactory MS/
MS spectra for both peptides were observed at 4 and
10 fmol levels using the SMW method for sample preparation and 400 laser shots. At the 1 fmol level, most fragment
ions of the precursor at m/z 1296.68 could still be observed
but only one from the larger peptide. Similar experiments
conducted with sample loading by the TL and DD methods
did not result in acceptable MS/MS spectra with the same
number of laser shots (data not shown). In general considerably higher laser energy was needed to obtain good
MS/MS spectra with the DD method than with the SMW
method, whereas all the sample would be depleted with
laser energies above threshold for the TL method. This
makes a direct quantitative comparison of the MS/MS performance of the three methods difficult. The data presented
above are obtained under conditions optimized to obtain the
best MS/MS spectra for each of the sample preparation
methods.
3.5 Oxidation in SMW method
Oxidation of methionine and tryptophan frequently occurs
during sample preparation for MALDI [4, 11, 13]. These oxidation reactions generate several oxidation products resulting in multiple mass signals for each peptide and consequently to reduce the protein identification efficiency. With
the SMW method, the peptide samples are exposed to air for
more than 10 min and therefore the risk of oxidation is high.
As shown in Fig. 6 for a 10 fmol/mL sample, almost all
methionine or tryptophan containing peptides were fully
oxidized resulting in decrease of the peak intensity of the
signal for the non-oxidized peptide. DTT is a well-known reducing reagent and can reduce oxidized methionine. Addition of DTT in the sample solution did indeed prevent oxidation of methionine but not of tryptophan (data not shown).
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Figure 4. Sample depletion upon multiple laser shots. 10 fmol pepmix was loaded on the target. (A) SMW method, from top to bottom,
spectra were collected from 1st, 3rd, 10th and 15th series of 50 shots. (B) DD method, from top to bottom, spectra were collected from 1st,
2nd, 6th and 7th series of 50 shots.
Figure 5. MS/MS spectra obtained with the SMW method for the precursor ions at m/z 2265.16 (A) and m/z 1296.68 (B). From top to bottom,
10 fmol, 4 fmol and 1 fmol loaded on the target.
N-octyl glucopyranoside (n-OGP), a nonionic detergent, was
reported to improve protein solubility [13]. Thus it should
also be expected to improve the embedment of the peptides
in the matrix and maybe also to shield the peptides for exposure to atmospheric oxygen during drying of the sample solution. This compound has previously been demonstrated to
be compatible with MALDI, and to enhance the responses
for high mass peptides [13, 17–19]. It was observed that the
oxidation of methionine and tryptophan could be considerably reduced if the peptide solution was made 2 mM
with respect to both n-OGP and DTT (Fig 6). This effect was
independent of the sample amount applied (data not shown)
and no significant change in signal intensity was observed
for peptides which did not contain methionine or tryptophan.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.6 Application of the SMW method in liver
proteomics
A total of 32 2-DE spots (Fig. 7) with varying spot intensities,
from a proteomics study of mouse liver, were submitted to
in-gel digestion and analyzed by MALDI-MS and MS/MS.
These digested solutions were loaded onto AnchorChipTM
targets using the three different methods. Acceptable peptide
mass fingerprints were obtained from nine spots using the
TL method and 24 respectively 32 spots using the DD and the
SMW methods. These PMF’s resulted in nine confidently
identified proteins with the TL method and 20 respectively
29 with the DD and SMW methods (Table 1). With the TL
method, it was not possible to obtain any MS/MS spectra of
reasonable quality. With the DD and the SMW methods good
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X. Zhang et al.
Proteomics 2007, 7, 2340–2349
Figure 6. The anti-oxidation effect obtained by adding n-OGP and DTT in the SMW method. The mass spectra were generated from loading
10 fmol pepmix with the SMW method without (upper spectrum) and with (lower spectrum) 2 mM n-OGP and 2 mM DTT in the sample
solution. The peak at m/z 1347 represents the non-oxidized form of Substance P (RPKPQQFFGL M-NH2) containing one methionine residue. The peak at m/z 3147 represents the non-oxidized form of Somatostatin 28 (SANSNPAMAP RERKAGCKNF FWKTFTSC) containing one
methionine and one tryptophan residue. The peaks marked with * represent the oxidized forms of peptides containing methionine and/or
tryptophan.
quality MS/MS spectra could be obtained from one or several peptide signals in most spectra. Typical MS and MS/MS
spectra from two different gel spots with the different
methods for sample preparation are shown Fig. 8. The protein in spot number 5 on the 2-DE (Fig. 8A) was identified
to be 60 kDa HSP based upon PMF and MS/MS data. The
TL, DD, and SMW methods resulted in 5, 24, and 27
matching peptides respectively. MS/MS spectra could be
only acquired by the DD and SMW methods resulting in
sequence confirmation and improved score in the MASCOT
search. The protein in spot number 4 on the 2-DE (Fig. 8B)
was identified to be selenium binding protein. Identifica© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
tion failed with the TL method due to too few peptide signals, whereas the MS spectra obtained with the SMW and
DD, resulted in significant protein identification. The identification data obtained for all the spots are summarized in
Table 1. Combining the PMF and MS/MS data obtained
with the DD and SMW methods, it was possible to significantly identify the proteins in 21 spots by DD and 31 spots
by SMW, whereas, identification was only possible from
nine spots on the basis of PMF data by the TL method. For
most of the spots, except three, the MASCOT score was
better for proteins identified from spectra recorded with the
SMW method.
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4
Figure 7. A representative 2-D gel image of mouse liver proteins.
The proteins (100 mg/gel) were separated on pre-cast IPG strips
(18 cm, pH 3210) in the first dimension and SDS-PAGE (12.5%) in
the second dimension followed by silver staining as described in
Section 2. pI and molecular mass standards are indicated on the
top and right side, respectively. The spots excised and submitted
for protein identification by MALDI-TOF/TOF are labeled with
arrows.
2347
Concluding remarks
A series of experiments were performed to compare the
three different methods for sample preparation on
AnchorChip™ targets. The SMW method presented here
provides better quality mass spectra and improved sensitivity
in MS as well as MS/MS mode compared to the previously
published methods. It also resulted in identification of more
proteins when tested in a mouse liver proteome project. For
the proteins identified using PMF data, the SMW method
resulted in more identified proteins and in most cases in a
higher score. Inclusion of MS/MS further increased the
score and allowed identification of two additional proteins in
the case of the SMW preparation method. Moreover, the
SMW method should be amenable for automation because it
does not involve pre-purification and pre-concentration steps
and because the washing procedure can be performed as a
batch process for the entire target. The SMW method for
sample preparation on AnchorChip™ targets has now been
used routinely for almost two years in our laboratory. The
performance described above has been reproducible. However, we have found that the addition of DTT and n-OGP was
not needed in routine protein identification because the
obtained improvement in score was only marginal.
Figure 8. MS and MS/MS spectra obtained from 2-DE spots number 5 (A) and 4 (B). The tryptic digests of these spots were loaded onto the
AnchorChip™ target with three different methods, respectively. The selected precursor ions are marked with * in the MS spectra.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 1. Identification of mouse liver proteins by MALDI-MS using the three different methods to load the samples onto the AnchorChipTM
target
Spot
Gi number
number
1
2
3
4
5
6
7
8
9
10
11
12
13
gi)115704
gi)115704
gi)130232
gi)22164798
gi)51702252
gi)5915682
gi)74186734
gi)5915682
gi)2498920
gi)52783095
gi)22653628
gi)30172916
gi)55154587
14
gi)21759113
15
16
17
18
19
20
21
23
24
gi)55976615
gi)416677
gi)232203
gi)121716
gi)3041732
gi)3219774
gi)5915682
gi)21362640
gi)51765519
25
26
27
28
29
30
31
32
gi)134614
gi)53237015
gi)1352250
gi)13637776
gi)117502
gi)56405010
gi)55154587
gi)30581036
Protein identification
Score from MASCOT
TL
PMF
Catalase
Catalase
Protein disulfide-isomerase A3 precursor
Selenium binding protein
60 kDa heat shock protein
Serum albumin precursor
unnamed protein product
Serum albumin precursor
Senescence marker protein-30
Inorganic pyrophosphatase
Alcohol dehydrogenase
Fructose-bisphosphate aldolase B
Similar to glyceraldehyde-3-phosphate
dehydrogenase
Electron transfer flavoprotein alpha-subunit,
mitochondrial precursor
Glycine N-methyltransferase
ATP synthase alpha chain, mitochondrial precursor
Glutathione S-transferase Yc
Glutathione S-transferase Mu 1
Superoxide dismutase [Mn], mitochondrial precursor
Peroxiredoxin 6
Serum albumin precursor
Lactoylglutathione lyase
PREDICTED: similar to hippocampal cholinergic
neurostimulating peptide precursor protein
Superoxide dismutase
Peptidylprolyl isomerase A
Aldehyde dehydrogenase, mitochondrial precursor
Alpha-enolase (2-phospho-D-glycerate hydro-lyase)
Calreticulin precursor
Elongation factor 1-alpha 1
Similar to glyceraldehyde-3-phosphate dehydrogenase
Carbonic anhydrase III
This work was supported by Chinese State Key Project for
Basic Research (No. 2004CB520802). Xumin Zhang was supported by a Ph.D. fellowship from Danish Research Councils. The
work was also part of the activities of the Danish Biotechnology
Instrument Center supported by the Danish Research Councils.
60
124
96
72
71
103
82
64
DD
PMF
PMF
Plus MS2
PMF
PMF
Plus MS2
65
80
80
65
186
116
137
109
137
128
96
216
116
181
129
192
160
232
109
109
248
113
186
197
224
144
208
91
181
106
171
118
142
260
167
242
239
320
184
250
132
224
156
219
163
174
145
186
191
231
99
99
69
73
78
173
108
73
78
208
114
122
152
182
127
139
161
130
155
157
187
229
168
184
204
176
60
60
60
80
60
80
60
61
65
79
126
251
130
123
169
294
172
75
172
199
149
65
68
SMW
126
162
110
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