Magnetic Chromatography brochure - Invitrogen

New
Magnetic Bead
Chromatography
Product Line
... including Dynabeads® RPC 18
DYNAL
invitrogen bead separations

Reduce sample complexity - no columns required!
Enjoy the benefits of Dynabeads®
The new magnetic bead chromatography product line is
intended for isolation of proteins and peptides in complex
samples such as serum, plasma, urine or cell lysate. The
products help you to:
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Reduce sample complexity
Concentrate and fractionate
Remove unwanted salts and contaminants
Perform serum profiling
Automate your analysis
Scale your protocols
Avoid columns or centrifugation
Produce reproducible results
Work with small amounts of starting material
Work with viscous samples
Biomarker discovery analysis needs
reproducible protocols
Biomarker discovery is a challenging field where
proteomic strategies are utilized for reliable detection of
disease-specific protein and peptide biomarkers in body
fluids. Discovery and validation of biomarkers require
large numbers of patient samples. Standardized and
automated methods generating reproducible results
are important, and Dynabeads® offer you a reliable high
throughput sample preparation method.
Simple magnetic bead chromatography
Magnetic chromatography Dynabeads® simplify sample
preparation of complex samples and offers a gentle
separation process, with no need for centrifugation or
columns (Fig. 1). Highly reproducible isolation allows for
the efficient recovery of proteins and peptides in small
volumes.
Dynabeads® RPC Protein
Dynabeads® RPC (Reversed Phase Chromatography)
Protein are perfectly suited for serum profiling of proteins
and large peptides (> 5,000 Da) and are ideal for top-down
proteomics strategies (Fig. 6). The amount of exposed
hydrophobic amino acids differs between proteins. The
more hydrophobic amino acids exposed, the stronger
is the binding to the surface of the beads. A step-wise
increase in acetonitrile concentration results in eluted
fractions of peptides/proteins (Fig. 3).
DYNAL
invitrogen bead separations
No Columns Required
Dynabeads® RPC 18
Dynabeads® RPC 18 are intended for peptide (< 5,000
Da) sample concentration, desalting (Fig. 5) and for the
reduction of sample complexity. The beads can be used
for serum profiling and peptide mass fingerprinting.
Peptides are desorbed from the beads using acetonitrile.
Fractionation of the peptide sample can be done
by eluting step-wise using increasing acetonitrile
concentrations.
Dynabeads® WCX
Dynabeads® WCX (Weak Cation Exchange) separate
proteins or peptides by exploiting differences in net
charge, where bead-bound negatively charged groups
adsorb positively charged molecules. Reduced sample
complexity by fractionation is brought about by
manipulating the pH or the salt concentration during
adsorption or elution (Fig. 2). Dynabeads® WCX can also
be used for serum profiling experiments (Fig. 6).
Automated sample preparation pre MS
analysis
Dynabeads® are optimal for automated applications
due to their small size (1 µm), low sedimentation rate
and high magnetic mobility. Magnetic separation and
handling can be automated on a wide variety of platforms.
Protein/peptide fractions obtained using magnetic
chromatography Dynabeads® can be applied to Matrix
Assisted Laser Desorption Ionisation (MALDI) targets for
MS analysis, allowing you to obtain excellent quality mass
spectra (Fig. 3, 4, 5 & 6) or analyzed in other downstream
applications such as electrospray-MS, HPLC or 1D/2D gel
electrophoresis (Fig. 2 & 3).
1

Magnetic Bead Chromatography
Fig. 1. Principle for magnetic bead chromatography products is very simple
The Dynabeads® are added to a protein/peptide sample and the proteins/peptides are allowed to adsob. The beads with bound material are
washed to remove contaminants (e.g. salt) by washing and magnetic separation. Reversed phase separation using Dynabeads® RPC 18 and
Dynabeads® RPC Protein is based on hydrophobic interactions, while Dynabeads® WCX utilise differences in net charge as the separating factor.
DYNAL
invitrogen bead separations
2

A
C
A
pH 3
Lysate (unfractionated)
655 spots
pH 3
400 mM fraction
Reduced Sample Complexity
802 spots, 68 % unique to this fraction 817 spots, 84 % unique to this fraction
DYNAL
invitrogen bead separations
pH 10
pH 10
B
D
pH 3
200 mM fraction
867 spots, 84 % unique to this fraction
pH 3 pH 10
600 mM fraction
Fig. 2. Reduced sample complexity using Dynabeads® WCX
A cell lysate from a human cancer cell line (SW480) was fractionated using Dynabeads® WCX. Proteins were adsorbed to Dynabeads® WCX using low salt conditions
(50 mM NaP buffer pH 7, 50 mM NaCl) and desorbed step-wise with increasing salt concentrations (50 mM NaP buffer pH 7 including 200 mM, 400 mM or 600 mM
NaCl). The figure shows unfractionated lysate (A) and the three fractions (B,C,D) as analysed by 2-D gel electrophoresis. Fraction B, C and D enabled the detection of
2,211 unique resolved spots and of these only 38 spots were found to be present in all three fractions (2%). Analysing the same amount of protein from unfractionated
lysate enabled the detection of only 655 spots.
Fractions were prepared for gel analysis by desalting using a commercially available 2-D gel clean up kit. Samples were then focused in pH 3 - 10 (11 cm) IPG strips
and resolved in 8 - 16 % SDS-PAGE gels. Approximately 100 µg protein was applied to each strip. Gels were silver stained and analysed using a commercially available
2-D gel analysis software.
Fig. 3. Reduced sample complexity using Dynabeads® RPC Protein
A. Fractionation of a protein mixture containing diamine oxidase and alcohol dehydrogenase using Dynabeads® RPC Protein. The protein mixture (containing 10 μg
total protein) was adsorbed to Dynabeads® RPC Protein in 200 mM NaCl, 0.1 % TFA. Salts were subsequently washed away and proteins desorbed step-wise using
increasing concentrations of acetonitrile (20 %, 30 % and 40 %). One quarter of each fraction was applied to a 10-20 % SDS-PAGE gel. The gel was silver stained.
Diamine oxidase and alcohol dehydrogenase are found in distinct fractions.
B. A mixture containing Insulin (1), Ubiquitin I (2), Cytochrome C (3) and Myoglobin (4) (Bruker Daltonics’ Protein Calibration Standard I) was fractionated using
Dynabeads® RPC Protein. The mixture was adsorbed to Dynabeads® RPC Protein in 200 mM NaCl, 0.1 % TFA. Salts were subsequently washed away and proteins/
peptides desorbed step-wise using increasing concentrations of acetonitrile (15 %, 30 % and 40 %). An aliquot of protein/peptide mixture or eluate (1 μl) was mixed
with an equal amount of MALDI matrix (1 μl sinapinic acid) and 0.5 μl of the sample/matrix mixture spotted onto a MALDI target plate. Data was obtained using a
commercially available MALDI-TOF/TOF MS instrument.
B
Intens. (a.u.)
Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
4
x10
3
2
1
0
x104
2.5
2.0
1.5
1.0
0.5
0 0
x104
2.0
1.5
1.0
0.5
0 0
4000
3000
2000
1000
0
1
pH 10
Unfractionated mixture
Pro1_g7_160305\0_G7\1\1SLin Raw
2 3 4
Pro2_g8_160305\0_G8\1\1SLin Raw
15 % acetonitrile fraction
Pro3_h6_160305\0_H6\1\1SLin Raw
30 % acetonitrile fraction
Pro4_h8_160305\0_H8\1\1SLin Raw
40 % acetonitrile fraction
6000 8000 10000 12000 14000 16000 18000
m/z
3

Intens. (a.u.)
Intens. [a.u.]
Intens. [a.u.]
Intens. Intens. [a.u.] (a.u.) Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
1500
1000
500
0
x104
3
2
1
0
x104
1.00
0.75
0.50
0.25
x104
1.5
1.0
0.5
4
x10
2
1
8000
6000
4000
2000
0
6000
4000
2000
0
8000
6000
4000
2000
Serum Profiling and Desalting
Pro8_g9_160305\0_G9\1\1SLin Raw
Untreated sample
Pro6_i7_160305\0_I7\1\1SLin Raw
Sample desalted using
Dynabeads ® RPC Protein
6000 8000 10000 12000 14000 16000 18000
Fig. 4. Desalt your sample pre MS analysis using Dynabeads® RPC
Protein
A mixture of Insulin, Ubiquitin I, Cytochrome C and Myoglobin containing
a high salt concentration (2.2 M NaCl) was desalted using Dynabeads®
RPC Protein. The mixture was adsorbed to Dynabeads® RPC Protein, salts
were subsequently washed away and proteins/peptides desorbed in 50 %
acetonitrile. An aliquot of protein/peptide mixture or eluate (1 μl) was mixed
with an equal amount of MALDI matrix (1 μl sinapinic acid) and 0.5 μl of the
sample/matrix mixture spotted onto a MALDI target plate. Data was obtained
using a commercially available MALDI-TOF/TOF MS instrument.
ser2_i4_LMW_040505\0_I4\1\1SLin Dynabeads Raw
® RPC Protein
ser4_j3_LMW_040505\0_J3\1\1SLin Dynabeads Raw
® RPC Protein
ser5_j5_LMW_040505\0_J5\1\1SLin Dynabeads Raw
® RPC Protein
Ser1_l20_110505\0_L20\1\1SLin Dynabeads Raw
® WCX
Ser2_m18_110505\0_M18\1\1SLin Dynabeads Raw
® WCX
Ser5_n19_110505\0_N19\1\1SLin Dynabeads Raw
® WCX
00 5000 6000 7000 8000 9000
m/z
m/z
A
B
Intens. (a.u.)
R4_050805_dig buff_TA_L8_100805\0_L8\1\1SRef Raw
Fig. 6. Dynabeads® RPC Protein and Dynabeads® WCX can be used
for Serum/Plasma Profiling to look for protein (and large peptide)
biomarkers
A single serum sample was divided into six identical aliquots (10 μl), to which
1 mg of Dynabeads® RPC Protein or Dynabeads® WCX and the appropriate
adsorption buffers were added. After washing, proteins (and peptides) were
eluted with either 50 % acetonitrile (Dynabeads® RPC Protein) or 0.5 % TFA
(Dynabeads® WCX). The results show that even using this non-automated
protocol sample preparation is reproducible. Both high and low abundant
protein peaks are detected which are above general instrument noise. Since
Dynabeads® RPC Protein or Dynabeads® WCX generate different profiles,
these beads can be used in parallel on the same serum samples in order to
increase the chance of identifying novel biomarkers.
An aliquot of eluate (1 μl) was mixed with an equal volume of MALDI matrix
(1 μl sinapinic acid) and 0.5 μl of the sample/matrix mixture spotted onto
a MALDI target plate. Data was obtained using a commercially available
MALDI-TOF/TOF MS instrument.
DYNAL
invitrogen bead separations 4
Intens. [a.u.]
Intens. [a.u.]
5
x10
1.0
0.8
0.6
0.4
0.2
0 0
x105
1.0
0.8
0.6
0.4
0.2
Digest spotted diectly
onto MALDI target
Pep41_dig buff_E810_TA_G5_100805\0_G5\1\1SRef Raw
Digest spotted after clean-up
with Dynabeads ® RPC 18
0 0
750 1000 1250 1500 1750 2000 2250 2500 2750
Contaminant Measurement Spotting after
Clean-up using
Dynabeads ® 6 M Urea
RPC 18
Fig. 5. Dynabeads® RPC 18 efficiently remove contaminants from peptide
samples
As a means of demonstrating desalting efficiency a tryptic Bovine Serum
Albumin (BSA) digest (2.5 pmoles) containing 6 M urea, 6 M guanidine, 2 M
NaCl or Digestion Buffer* (50 mM NH 4 HCO 3 , 0.5 M urea, 1 mM DTT, 0.1 % TFA)
was desalted using Dynabeads® RPC 18. The peptide mixture was adsorbed
to Dynabeads® RPC 18, salts were subsequently washed away and peptides
desorbed in 50 % acetonitrile. An aliquot of digest or eluate (1 µl) was mixed
with an equal amount of MALDI matrix (1 µl HCCA) and 0.5 µl of the sample/
matrix mixture spotted onto a MALDI target plate. Data was obtained using
a commercially available MALDI-TOF/TOF MS instrument. An example of
typical spectra are shown (A) and the table (B) summarises the efficiency
of desalting as demonstrated by sequence coverage, Mascot® Scores and
numbers of peptides matched during database searching (n = 6).
m/z
Direct Spotting
onto a
MALDI Target
Sequence Coverage (%) 35 ± 1 No identification
Mascot ® Score 199 ± 7 No identification
Peptides Matched 19 ± 1 No identification
Sequence Coverage (%) 25 ± 2 No identification
Mascot ® 6 M Guanidine
Score 142 ± 10 No identification
Peptides Matched 14 ± 2 No identification
Sequence Coverage (%) 30 ± 8 9 ± 1
Mascot ® 2 M NaCl
Score 170 ± 52 37 ± 8
Peptides Matched 16 ± 4 5 ± 1
Sequence Coverage (%) 40 ± 3 No identification
Mascot ® Digestion
Buffer*
Score 238 ± 19 No identification
Peptides Matched 22 ± 2 No identification

Ordering Information
Product Concentration Volume Product No.
Dynabeads® RPC 18 50 mg / ml 2 ml 102.01
Dynabeads® RPC 18 50 mg / ml 10 ml (5 x 2 ml) 102.02
Dynabeads® RPC Protein 50 mg / ml 2 ml 102.06
Dynabeads® RPC Protein 50 mg / ml 10 ml (5 x 2 ml) 102.07
Dynabeads® WCX 50 mg / ml 2 ml 105.01
Dynabeads® WCX 50 mg / ml 10 ml (5 x 2 ml) 105.02
...more products to come!
© Copyright 2005 Dynal Biotech ASA, Norway. Printed 09.05. All rights reserved.
Dynal ® , Dynabeads® and Dynal MPC® are registered trademarks of Dynal Biotech ASA.
The Dynabeads® products are covered by several international patents and patent applications.
Dynal is an ISO 9001 certified company and is part of Invitrogen Corporation.
Dynal will not be responsible for violations or patent infringements that may occur with the use of our products.
DYNAL
invitrogen bead separations
BBS.B.002.01
www.invitrogen.com
www.dynalbiotech.com