Instructions for Use - Glyco Kit MB-LAC Con A - Bruker

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Instructions for Use
Glyco Kit MB-LAC Con A
As part of Bruker Daltonics’ CLINPROT system the Glyco Kits are especially designed for
sophisticated glycoprotein studies and clinical proteomics research. Super-paramagnetic glycobeads
are functionalized with either boronic acids (BA) or lectins like concanavalin A (Con A),
wheat germ agglutinin (WGA), Lens Culinaris Agglutinin (LCA), artocarpus integrifolia (AIA or
Jacalin). With a combination of different optimized beads, intelligent enrichment strategies for
glycosylated peptides and proteins prior to mass spectrometry enable advanced glycomic
analyses. With the Bruker Daltonics’ mass spectrometry workflow, glycoproteins from complex
samples like human serum can be reliably and reproducibly captured and identified within a
dynamic range of eight orders of magnitudes. Besides glycoprotein identification and
characterization as well as glyco-structure predictions, CLINPROT glyco-beads are suitable for
sample fractionation prior to profiling analyses.
Sample
Bruker Daltonics CLINPROT magnetic glyco-beads
Separation on protein level
(e.g. ConA, LCA, WGA, AIA or BA)
LC-MALDI
Tryptic digestion
CLINPROT TM bead
purification
(e.g. C8, WCX, IMAC-Cu)
Bruker Daltonics CLINPROT magnetic glyco-beads
Separation on peptide level
(ConA)
PNGase F digestion
LC-MALDI
LC-MALDI
Identification of
glycoproteins
Structural characterization
of N-glycosylated peptides
Identification of
N-glycosylation sites
Glyco-Profiling
Fig. 1: Range of possible experimental strategies for the application of Bruker Daltonics’ CLINPROT TM
magnetic glyco-beads.
Page 1 of 21

Figure 1 exemplarily depictured possible experimental strategies to facilitate the identification of
glyco-proteins, to enable the detection of N-glycosylation sites, to support the structural
characterization of N-glycosylated peptides and to perform glyco-profiling studies in the clinical
context.
General Information
Glycosylation is the most common post-translational modification of proteins, playing a key role in
protein folding, stability, solubility, activity and molecular recognition. Altered glycosylation is known
to act as an indicator or effector in pathologic mechanisms with potential impact on diagnosis and
therapy of human diseases. Immunity to certain infectious diseases, prion diseases and cancer
can all be associated with abnormal glycosylation patterns. As an example, increased branching
and sialylation of N-linked glycans on cancer cells are potential tumor markers in molecular
diagnostics.
Bruker Daltonics’ CLINPROT magnetic glyco-beads, specifically functionalized with lectins or
boronic acids that bind to various structural motifs, facilitate the selective isolation of different
glycoproteins out of complex biological sources (Fig. 1). Bruker Daltonics has developed workflows
combining the specific pre-fractionation employing glyco-specific magnetic particles, e.g. ConA,
WGA, LCA or boronic acids coated beads, and LC-MALDI analysis to identify glycoproteins, to
locate N-glycosylation sites and to get structural information about N-glycosylated peptides.
MB-LAC
Con A
MB-LAC
LCA
MB-LAC
WGA
MB-LAC
AIA
MB-CovAC
boronic
High-mannose type Hybrid type Complex type
Glycorotein
Glycoprotein
Glycoprotein
Man
GlcNAc
Gal
Fuc
NeuAc
GalNAc
Glycoprotein
Glycoprotein
Glycoprotein
O-linked glycans
C-linked glycans
N-linked glycans
Fig. 2: Binding motifs of Bruker Daltonics’ CLINPROT TM
represent overlapping but individual binding profiles.
magnetic glyco-beads. The different beads
Successful applications and reproducibility of Bruker Daltonics’ CLINPROT TM magnetic glycobeads
using model systems or complex samples like serum, plasma or urine were demonstrated in
various studies 1,2,3 . Further, long term stability and storage conditions were extensively
investigated to assure the high quality of Bruker Daltonics’ CLINPROT TM magnetic glycobeads
4 .The application of glyco-beads in the context of glyco-profiling and biomarker discovery
was closely described by Drake et al. 5
Page 2 of 21

Table of Contents
General Information........................................................................................................................... 2
Intended Use..................................................................................................................................... 4
Product description............................................................................................................................ 4
Kit Components................................................................................................................................. 5
Storage and lifetime information........................................................................................................ 5
Risk and safety information ............................................................................................................... 5
General remarks for sample handling and MALDI measurements ................................................... 6
Recommended sample preparation procedure ................................................................................. 7
Comments ....................................................................................................................................... 10
MALDI-TOF MS Target Preparation................................................................................................11
Cleaning of MALDI-MS targets.................................................................................................... 11
Cleaning of AnchorChip TM targets ........................................................................................... 11
Cleaning of polished steel targets ........................................................................................... 12
Matrix preparation on the MALDI-MS targets.............................................................................. 13
Analysis of glycoproteins using 2,5-DHAP .............................................................................. 13
Analysis of glycopeptides using 2,5-DHB................................................................................ 14
Analysis of deglycosylated peptides using PAC targets.......................................................... 16
Examples......................................................................................................................................... 17
Isolation of a small glycoprotein from a protein mixture .............................................................. 17
Isolation of glycopetides from tryptic RNase B digest ................................................................. 18
References...................................................................................................................................... 19
Trouble shooting.............................................................................................................................. 20
Ordering Information ....................................................................................................................... 21
Support............................................................................................................................................ 21
Page 3 of 21

Intended Use
The Glyco Kit MB-LAC Con A (Magnetic Bead-Lectin Affinity Chromatography with Concanavalin
A) is developed for specific capturing of N-glycosylated proteins and peptides derived from
biological samples based on lectin affinity prior to MALDI-TOF mass spectrometry analysis by
means of e.g. Bruker´s microflex ® , autoflex ® or ultraflex ® MALDI-TOF.
This kit is for research use only. It is not for use in diagnostic procedures.
Product description
The MB-LAC Con A kit is designed for enrichment and purification of N-glycosylated peptides and
proteins prior to MALDI-TOF MS analysis. The MB-LAC Con A kit is based on super-paramagnetic
microparticles functionalized with Concanavalin A.
Concanavalin A is a lectin which specifically binds glucose and/or mannose residues of
polysaccharides and glycoprotein/-peptides containing unmodified hydroxyl groups at positions C3,
C4 and C6 6 , 7 , 8 . The binding motif is depicted in Fig. 3. The green highlighted mannose residues of
the pentacore of N-glycosylated peptides/proteins interact with Concanavalin A.
MB-LAC
Con A
Glycoprotein
Man
GlcNAc
Fig. 3: Pentacore of N-glycosylated peptides/proteins. Mannose
residues responsible for the interaction with Concanavalin A
are highlighted in green.
Due to the binding requirements ConA binds N-glycans of the high mannose type, the hybrid type
and bi-and tri-antennary complex type. According to this, it can be applied as a general tool for the
capturing of N-glycosylated peptides and proteins with broad specificity.
Page 4 of 21

Kit Components
Materials provided in the kits Abbreviation Remarks
Magnetic Beads MB-LAC Con A Containing 0.02 % NaN 3
MB-LAC Con A Binding Buffer 1 BB 1
MB-LAC Con A Binding Buffer 2 BB 2
MB-LAC Con A Wash Buffer 1.1 WB 1.1
MB-LAC Con A Wash Buffer 2.1 WB 2.1
MB-LAC Con A Wash Buffer 2 WB 2
MB-LAC Con A Elution Buffer
EB
Deionized water
Storage and lifetime information
Kit components should be stored and handled carefully to avoid contaminations.
The kit is shipped at ambient temperature. We recommend storing the kit at 2-8°C after arrival. Do
not freeze!
If stored unopened under above conditions, the product is stable until expiration date mentioned on
the label.
Please note:
After the first opening, period of storage should not exceed three months. For your
convenience, we provide the possibility to mark the respective expiry date on the vial’s label.
After usage, keep the vials closed. A drying up of the beads will damage the product
irreversible!
Risk and safety information
The product does not have to be labeled due to the calculation procedure of the "General
Classification guideline for preparations of the EU" in the latest valid version. When used and
handled according to specifications, the product does not have any harmful effects according to
our experience. All materials may present unknown hazards and should be used with caution.
Observe the general safety regulations when handling chemicals.
Beside the kit components, we recommend further chemicals within these Instructions for Use.
Please read and observe the respective Material Safety Data Sheet to be provided by your
supplier. Observe the general safety regulations when handling chemicals.
Page 5 of 21

General remarks for sample handling and MALDI measurements
MALDI-TOF mass spectrometry is a very sensitive analysis method requiring special operation
procedures. Therefore, we recommend considering the following aspects before starting an
analysis:
The quality of the starting material will have an enormous impact on the outcome of the
experiments. Especially, collection and storage conditions have to be controlled carefully.
When working with clinical samples, like serum or urine, please refer to the recent
publications of Baumann et al. 9 and Fiedler et al. 10 for standardized sample handling
procedures.
To avoid contaminations all solvents used should be of HPLC grade or better
The plastic materials used for sample purification and matrix preparation should be
explicitly recommended for mass spectrometric measurements. Many common plastics
may cause polymer contamination of the sample. This will interfere with the subsequent
matrix preparation/crystallization and produce polymer signals during spectra acquisition.
Sample purification and MALDI target preparation should be performed under clean
conditions. Dust and air pollution will decrease the spectra quality.
For optimal sample/matrix crystallization on the MALDI target the following environmental
conditions are recommended:
Operating temperature: 5 - 40°C (41 - 104°F)
Operating humidity: 25 – 65 % at 22°C
Atmospheric pressure: 75 - 105 kPa
For high resolution measurements and low signal to noise values a cleaning of the ion
source at regular intervals is absolutely mandatory. This procedure can be performed with
the Bruker Daltonics’ source shower target (see ordering information). For a complete
cleaning of the ion source please ask Bruker Daltonics’ service team
(maldi.support@bdal.de).
Page 6 of 21

Recommended sample preparation procedure
Important: The sample should not contain any Ca 2+ , Mg 2+ and Mn 2+ chelating agents, e.g. EDTA.
The isolation of glyco-proteins and glyco-peptides requires different buffer systems.
For the isolation of glyco-proteins please use:
BB 1
WB 1.1.
WB 2
EB
For the isolation of glyco-peptides please use:
BB 2
WB 2.1.
WB 2
EB
Binding of samples
1. Re-suspend the Magnetic Beads by shaking
for at least 20 times from top to the bottom
and reversed or careful vortexing (do not
sonificate!) to get a homogenous suspension.
Repeat shaking between pipetting steps if
necessary.
Re-suspend the beads
2. Transfer 20 µl of resuspended Magnetic
Beads to a standard thin wall PCR-tube and
add 100 µl WB 1.1 (for glyco-proteins) or
WB 2.1 (for glyco-peptides).
20 µl beads
100 µl WB1.1 or WB2.1
3. Place the tube in a Magnetic Separator and
move the tubes back and forth between
adjacent wells 20 times. Wait for 20 seconds
to separate the beads from the supernatant.
Move back and forth
Collect beads
4. Remove the supernatant carefully using a
pipette. Avoid contact of pipette tips with the
magnetic beads and take care not to remove
the beads.
Remove supernatant
5. Resuspend the beads in 100 µl WB 1.1 or WB
2.1.
100 µl WB1.1 or WB2.1
Page 7 of 21

6. Repeat steps 3 and 4 to wash the beads and
remove the supernatant
Repeat steps 3 and 4
7. Re-suspend the beads in 10 µl BB 1 (for
glyco-proteins) or BB 2 (for glyco-peptides)
and add up to 10 µl of the sample. If you have
less sample volume adjust to 10 µl by adding
deionized water.
For the analysis of serum, use 10 - 20 µl
serum. Firstly, pipette an equal amount of BB
1 or BB 2 (10 - 20 µl) to 20 µl magnetic beads
and subsequently add the serum.
10 µl BB1 or BB2
10 µl sample
8. Incubate the magnetic beads for 1 h at room
temperature while swirling the tube from time
to time to mix the beads.
1 h incubation
Washing of beads
9. Place the tube on the Magnetic Separator and
wait for 20 seconds to separate the beads
from the supernatant. (Note the movement of
the magnetic beads in the tube.)
Collect beads
10. Remove the supernatant carefully. Avoid
contact of pipette tips with the magnetic beads
and take care not to remove the beads.
Remove supernatant
11. Add 100 µl WB 1.1 (for glyco-proteins) or
WB 2.1 (for glyco-peptides).
100 µl WB1.1 or WB2.1
12. Move the tubes back and forth between
adjacent wells at least 20 times to wash the
beads.
Move back and forth
13. Wait 20 seconds for collecting magnetic beads
at the wall of the tubes and remove the
supernatant carefully.
Collect beads
Page 8 of 21

14. Transfer the tube from the Magnetic Separator
to an alternative device. Add 100 µl WB2 and
place the tube back on the Magnetic
Separator. Move the tubes back and forth
between adjacent wells 20 times.
100 µl WB2
Move back and forth
15. Wait 20 seconds for collecting magnetic beads
at the wall of the tubes and remove the
supernatant carefully.
Collect beads
Remove supernatant
16. Repeat steps 14 and 15 once. Repeat steps 14 and 15
Elution of isolated peptides/proteins
17. For the elution of captured peptides and
proteins add 10 µl EB and mix thoroughly.
10 µl EB
18. Incubate for 25 minutes while mixing from time
to time.
25 min incubation
19. Place the tube on the Magnetic Separator and
wait for 30 seconds for collecting the beads at
the wall of the tube.
Collect beads
20. Finally, transfer the elution solution containing
purified glycopeptides/-proteins into a fresh
tube.
Transfer eluate
Page 9 of 21

Comments
Fast and successful collection of the magnetic beads is dependent on a high quality magnetic
separation device. We strongly recommend the use of a Bruker Magnetic Separator (8-well,
# 65554 or 96-well, # 207151).
Alternatively, elution can be performed with 50 % acetonitrile in 1 % TFA. We strongly recommend
using Acetonitrile hypergrade quality for liquid chromatography. Prepare the solution daily.
: Acetonitrile is highly flammable and harmful (R: 11-20/21/22-36; S: (1/2)-16-36/37), 2% TFA
is irritant (R: 36/38; S: 26-28-37-60)
The beads can also be applied for non-MS approaches. Depending on the subsequent technique
the acidic elution solution may interfere with the following protocol steps. To remove the elution
solution we recommend drying the eluate in a vacuum centrifuge. Afterwards, the dried sample can
be re-dissolved in an appropriate buffer or solution and processed according to the subsequent
protocol. This procedure is also recommended when different glyco-capturing particles are used in
succession.
Different glyco-beads can also be combined in one experiment to capture glyco-proteins with
different binding motifs at once. To do so, the buffer system of either a ConA or LCA kit has to be
used since these buffer systems are compatible with the WGA and/or AIA beads, whereas the
buffers in the WGA and AIA kits are not suitable for the ConA or LCA functionality.
In principle, the amount of beads can be scaled up and down according to the experimental
requirements. When doing this, it is important to keep the ratio of sample volume and ConA-BB
volume constant.
Please note:
After elution using the provided EB, glyco-proteins are denatured. This will have an impact on
folding and functional activity.
Page 10 of 21

MALDI-TOF MS Target Preparation
MALDI-TOF MS target preparation is one of the most critical steps for high quality and reproducible
results. Depending on the application, different target types and MALDI matrix preparations are
favored. For all applications it is mandatory to use a thoroughly cleaned target plate. In
dependence on the type of target Bruker Daltonic recommends different cleaning procedures.
Cleaning of MALDI-MS targets
Cleaning of AnchorChip TM targets
This is an advanced cleaning protocol differing from the standard cleaning procedure
recommended in the product description.
1. Rinse the target intensively under
flowing hot tap water
Hot tap water
2. Wipe the target intensively with acetone
using a kimwipe kimwipe.
: Acetone is highly flammable and
irritant (R: 11-36-66-67; S: (2)-9-16-26)
Acetone
3. Rinse the target with distilled water
(Milli-Q).
Distilled water
4. Rinse the target with methanol and let it
dry.
: Methanol is highly flammable and
toxic (R: 11-23/24/25-39/23/24/25; S:
(1/2)-7-16-36/37-45)
Methanol
Page 11 of 21

Cleaning of polished steel targets
This is an advanced cleaning protocol differing from the standard cleaning procedure
recommended in the respective target’s product description.
1. Rinse the target intensively under
flowing hot tap water.
Hot tap water
2. Wipe the target intensively with acetone
using a kimwipe.
: Acetone is highly flammable and
irritant (R: 11-36-66-67; S: (2)-9-16-26)
Acetone
3. Rinse the target intensively under
flowing hot tap water.
Hot tap water
4. Wipe the target intensively with 80%
TFA using a kimwipe.
: 80% TFA is corrosive (R: 20-35-
52/53; S: (1/2)-9-26-27-28-45-61)
80 % TFA
5. Rinse the target intensively under
flowing hot tap water.
Hot tap water
6. Rinse the target with distilled water
(Milli-Q).
Distilled water
7. Rinse the target with methanol and let it
dry.
: Methanol is highly flammable and
toxic (R: 11-23/24/25-39/23/24/25; S:
(1/2)-7-16-36/37-45)
Methanol
Page 12 of 21

Matrix preparation on the MALDI-MS targets
Analysis of glycoproteins using 2,5-DHAP
For preparation of glycoproteins within the mass range of 6 – 100 kDa on AnchorChip TM 600
targets (#209513) we recommend the use of 2,5-dihydroxyacetophenone (2,5-DHAP, #231829 or
#231830) as matrix solution.
2,5-DHAP preparation on AnchorChip600 targets
1. Dissolve 7.6 mg DHAP in 375 µl
ethanol.
: DHAP is irritant (R: 36/37/38; S:
26-28-36/37-60), ethanol is highly
flammable (R: 11; S: (2)-7-16)
7.6 mg DHAP
375 µl ethanol
2. Add 125 µl (18 mg/ml in Milli-Q water)
of a di-ammonium hydrogencitrate
solution (= 10 µmol).
: Diammonium hydrogencitrate is
irritant (R: 36/37; S: 26)
125 µl Diammonium
Hydrogen Citrate
3. Vortex for 1 min at RT. Vortex
4. Sonicate for 15 min. Sonificate
5. Vortex for 1 min at RT. The matrix
solution is stable for at least one week
when kept in the dark
Vortex
6. Mix 2 µl of the sample with 2 µl 2 %
TFA and subsequently add 2 µl matrix
solution.
: 2% TFA is irritant (R: 36/38; S: 26-
28-37-60)
2 µl Sample
2 µl 2% TFA
2 µl DHAP Matrix Solution
7. The mixture of sample, TFA and DHAP
has to be vigorously mixed by pipetting
up and down. It is important that the
solution becomes turbid before spotting
Vigorously mixing
Page 13 of 21

8. 1 µl of the turbid suspension has to be
spotted on an AnchorChip TM 600 target
without generating air bubbles.
Spot 1 µl
9. After drying of the spots the target can
be transferred to the MALDI-TOF MS
for measurements.
Dry the spot
Analysis of glycopeptides using 2,5-DHB
For preparation of glycopeptides (e.g. after proteolytic digest of the glycoprotein) within the mass
range of 1 – 10 kDa we recommend the use of AnchorChip 600 targets (#209513) or polished
steel targets (#209520) and 2,5-Dihydroxybenzoic acid (DHB, #201346 or #203074) as matrix.
2,5-DHB preparation on AnchorChip600 targets
1. Dissolve 5 mg DHB in 300 µl
acetonitrile
: DHB is harmful (R: 22-36/37/38;
S: 26-28-36/37-60), acetonitrile is
highly flammable and harmful (R: 11-
20/21/22-36; S: (1/2)-16-36/37)
5 mg DHB
300 µl acetonitrile
2. Add 700 µl TFA (0.1 %) 700 µl 0.1 %TFA
3. Vortex for 1 min at RT Vortex
4. Sonificate for 15 min Sonificate
5. 1 µl of the freshly prepared matrix has to
be spotted onto an AnchorChip 600
target
Spot 1 µl matrix
6. After drying spot 1 µl of sample onto the
matrix
Spot 1 µl sample
Page 14 of 21

7. After drying of the spots the target can
be transferred to the MALDI-TOF MS for
measurements.
Dry the spot
2,5-DHB preparation on polished steel targets
1. Dissolve 10 mg DHB in 300 µl
acetonitrile
: DHB is harmful (R: 22-36/37/38; S:
26-28-36/37-60), acetonitrile is highly
flammable and harmful (R: 11-20/21/22-
36; S: (1/2)-16-36/37)
5 mg DHB
300 µl acetonitrile
2. Add 700 µl TFA (0.1 %) 700 µl 0.1 %TFA
3. Vortex for 1 min at RT Vortex
4. Sonificate for 15 min Sonificate
5. 1 µl of the freshly prepared matrix has to
be spotted onto an AnchorChip TM 600
target
Spot 1 µl matrix
6. After drying spot 1 µl of sample onto the
matrix
Spot 1 µl sample
7. After drying of the spots the target can be
transferred to the MALDI-TOF MS for
measurements.
Dry the spot
Page 15 of 21

Analysis of deglycosylated peptides using PAC targets
For the analysis of deglycosylated peptides (e.g. after PNGase F treatment) the use of PAC (Prespotted
AnchorChip) targets prespotted with α-Cyano-4-hydroxycinnamic acid (HCCA) is
recommended (#231968 or #227463).
Sample preparation onto pre-spotted disposable AnchorChip targets
1. Deposit 0.3 – 3 µl sample soluton onto
the matrix spot. The acetonitrile
concentration of the sample must not
exceed 40 %
Spot 0.3 – 3 µl sample
2. Incubate the sample for 3 min onto the
spot, but do not let the droplets dry
3 min incubation
3. Remove the residual solvent Remove droplet
4. Add 7 – 10 µl of washing buffer (10mM
Ammonium phosphate, monobasic, in
0.1 % TFA)
Wash with 7 – 10 µl
5. Incubate for a few second and
subsequently remove the wash buffer
Remove wash buffer
after a few seconds
Page 16 of 21

Examples
Isolation of a small glycoprotein from a protein mixture
Sample:
Protein mixture consisting of Protein Standard II (1 µl, #207234) and
RNase B (1 µg)
Beads:
MB-LAC Con A (10 µl)
Buffers:
BB 1, WP 1.1, WP 2, EB
Target: MTP AnchorChip 600/384
Matrix:
2,5-DHAP
MALDI mode:
Linear
1500
14920.6
66381.6
Original sample
1000
500
23981.6
44528.4
Intens. [a.u.]
0
1500
1250
1000
750
66399.8
Supernatant
14931.7 23987.7 44548.5
500
250
0
2000
1500
1000
14937.2
Eluate
500
44607.5
0
10000 20000 30000 40000 50000 60000 70000 80000 90000 m/z
RNase was successfully isolated by MB-LAC Con A beads from a protein mixture consisting of
BSA (66 kDa), Protein A (44 kDa) Trypsinogen (23 kDa) and RNase B (14.9 kDa).
Page 17 of 21

Isolation of glycopetides from tryptic RNase B digest
Sample:
Tryptic digest of RNase B (10 pmol)
Beads:
MB-LAC Con A (10 µl)
Buffers:
BB 2, WP 2.1, WP 2, EB
Target: MTP AnchorChip 600/384
Matrix:
2,5-DHB
MALDI mode:
Reflector
8000
2224.014
2760.078
Total digest
Intens. [a.u.]
6000
4000
2000
0
1200
1070.410
1150.567
1287.584
1399.618
1504.603
1691.612
1934.747
2123.800
2170.808
2286.860
2364.870
2421.885
2517.165
H H H H
2672.212
2712.085
2817.102
2896.080
3023.353
3241.313
3332.334
Eluate
1000
800
1934.837
2420.982
600
400
200
SRNLTK
1991.847
2096.882
2258.926
2364.905
2477.985
2583.029
0
1250 1500 1750 2000 2250 2500 2750 3000 3250 3500 m/z
The glycosylated peptide RNase B aa58-63 was successfully isolated from the complex total digest
of RNase B. The peak at 1934.8 Da correlates to the depictured glyco-peptide structure comprising
five mannose residues. Four additional signals each with a distance of 162 Da (one hexose unit) to
the previous peak correspond to higher mannosylated forms of the same peptide comprising six,
seven, eight or nine mannose residues, respectively.
Page 18 of 21

References
1 Sparbier K, Koch S, Kessler I, Wenzel T, and Kostrzewa M.(2005). Selective Isolation of
Glycoproteins and Glycopeptides for MALDITOFMS Detection Supported by Magnetic Particles. J
Biomol Tech, 16: 407 – 413.
2 Sparbier K, Wenzel T, Kostrzewa M. (2006). Exploring the binding profiles of ConA, boronic acid
and WGA by MALDI-TOF/TOF MS and magnetic particles. J ChromB, 840: 29–36.
3 Sparbier K, Asperger A, Resemann A, Kessler I, Koch S, Wenzel T, Stein G, Vorwerg L, Suckau
D, Kostrzewa M. (2007). Analysis of Glycoproteins in Human Serum by Means of Glyco-specific
Magnetic Bead Separation and LC-MALDI-TOF/TOF Analysis with Automated Glycopeptide
Detection. J Biomol Tech, Sept., accepted for publication.
4 Sparbier K; Kessler I, Koch S, Mix G, Kostrzewa M. (2007). CLINPROT Glyco-beads: Verification
of Long-Term Stability and Batch-to-Batch Reproducibility. Technote Bruker Daltonic (tn-20).
5 Drake RR, Schwegler EE, Malik G, Diaz J, Block T, Mehta A, and Semmes OJ. (2006). Lectin
Capture Strategies Combined with Mass Spectrometry for the Discovery of Serum Glycoprotein
Biomarkers. Mol&CellProt, 5.10:1957 – 1967.
6 Kamra A, GuptaMN. (1987). Crosslinked concanavalin A-O-(diethylaminoethyl)-cellulose--an
affinity medium for concanavalin A-interacting glycoproteins. Anal. Biochem., 164: 405 - 410.
7 Yahara I, Edelman GM. (1972). Restriction of the mobility of lymphocyte immunoglobulin
receptors by concanavalin A. PNAS, 69: 608 - 612.
8 Goldstein IJ, Hollerman CE, SmithEE. (1965). Protein-Carbohydrate Interaction. II. Inhibition
Studies on the Interaction of Concanavalin A with Polysaccharides. Biochemistry, 41: 876 -883.
9 Baumann S, Ceglarek U, Fiedler GM, Lembcke J, Leichtle A, Thiery J. (2005): Standardized
approach to proteome profiling of human serum based on magnetic bead separation and matrixassisted
laser desorption/ionization time-of-flight mass spectrometry. Clin Chem., 51, 973-80.
10 Fiedler GM, Baumann S, Leichtle A, Oltmann A, Kase J, Thiery J, Ceglarek U. (2007):
Standardized peptidome profiling of human urine by magnetic bead separation and matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry. Clin Chem. 53, 421-8.
Page 19 of 21

Trouble shooting
Problem Reason What to do
Sample does not crystallize,
crystallization looks
strange
Plastics (tips, tubes, flasks)
are not MS-compatible
Solvents are not of HPLC
grade or better
Environmental conditions
are not compatible with MS
preparations
Change plastics (referr to
the ording information for
MS compatible plastics
Exchange the solvents and
use higher quality solvents
Prepare under a clean
bench, check humidity,
maybe humidify the room,
switch off the air condition
Polymer contaminations
Plastics (tips, tubes, flasks)
are not MS-compatible
Solvents are not of HPLC
grade or better
Change plastics (refer to
the ordering information for
MS compatible plastics)
Exchange the solvents and
use higher quality solvents
Low signal/noise values Environmental conditions
are not compatible with MS
preparations
Air pollution and subsequent
contamination with
sodium or potassium
Prepare under a clean
bench, check humidity,
maybe humidify the room,
switch off the air condition
See above
Quality of the starting
material
No detection of glycoproteins/-peptides
Glyco-proteins/-peptides do
not represent the required
binding motifs
Try other glyco-beads
functionalized with another
lectins or boronic acids
beads
Page 20 of 21

Ordering Information
Product
Size (purifications or
applications per kit)
Part No.
Glyco Kit 20 MB-LAC Con A 20 252665
Glyco Kit 100 MB-LAC Con A 100 252664
Glyco Kit 20 MB-CovAC boronic 20 241104
Glyco Kit 20 MB-LAC WGA 20 244669
Glyco Kit 20 MB-LAC LCA 20 246534
Glyco Kit 20 MB-LAC AIA 20 250359
Glyco Kit 100 MB-CovAC boronic 100 236594
Glyco Kit 100 MB-LAC WGA 100 244668
Glyco Kit 100 MB-LAC LCA 100 246533
Glyco Kit 100 MB-LAC AIA 100 249174
Magnetseparator 016 8-well 65554
Magnetseparator 104 II 96-well 207151
2,5-Dihydroxyacetophenone, 1g 231829
2,5-Dihydroxyacetophenone, 5g 231830
2,5-Dihydroxybenzoic acid, 1 g 201346
2,5-Dihydroxybenzoic acid, 5 g 203074
α-Cyano-4-hydroxycinnamic acid, 1g 201344
α-Cyano-4-hydroxycinnamic acid, 5g 203072
MTP AnchorChip 600/384 T F 209513
PAC96 Set for Proteomics 231968
PAC384 Set for Proteomics 227463
MTP PAC Frame 221598
MTP 384 target plate polished steel T F 209520
MTP Ion Source Shower Target 226054
8-Well Strips 60550
CapStrips 60551
96-Well TubePlates 61565
Bruker Daltonics’ Recommendations
Acetonitrile, LC-MS Grade
Pipette tips
Vials
Bottles
e.g. Carl Roth GmbH & Co. KG
Eppendorf standard tips
Eppendorf safe-lock
Nalgene PP
Support
Email: care@bdal.de
Phone: +49 (421) 2205-0
Web: www.care-bdal.de
For research use only. Not for use in diagnostic procedures.
Revision 1, January 2012
Copyright 2012 Bruker Daltonik GmbH
Descriptions and specifications supersede all previous information and are subject to change without notice.
Page 21 of 21