NuGel™ NRicher™ Mx - Biotech Support Group

NuGel™ NRicher™ Mx (Formerly NuGel
PROfessor™)
Supports two applications:
Compound Centric Chemical Proteomics (CCDP) &
Protein Compression
NuGel™ NRicher™ Mx (formerly NuGel
PROfessor™) supports compound-centric
displacement proteomics (CCDP)




Non-covalently binds proteins, a subset of which
can be displaced upon introduction of soluble
small compounds
Coupled to LC-MS, spectral counts of affinityeluted pools can serve as quantitative metrics to
characterize and identify interacting proteins
Gain efficiencies over prior covalent-based
substitution methods: more complete survey, no
orientation bias, robust background controls
NuGel™ NRicher™ Mx (formerly NuGel
PROfessor™) can compress protein
concentrations, enrich low abundance
proteins

Function, structure and sequence can be
characterized and correlated

Low abundance proteome enriched 5-10X

Tissue and species agnostic

Suitable for small volume mg scale preps
 On-bead digestion protocols
 No pI or molecular weight bias
Applications in drug target deconvolution, ontarget/off-target specificity, and personalized
medicine.
MS2 Spectral Counts of CCDP-derived Sub-proteomes
Protein Description
Hemoglobin subunit beta-1
Glucose-6-phosphate isomerase
Malate dehydrogenase
transketolase
Cytochrome c, somatic
Succinyl-CoA:3-ketoacid transf
Transgelin
Annexin A2
fumarate hydratase
annexin A3
glutathione reductase
Caffeine
87
192
117
72
47
69
0
26
17
5
9
Imatinib
550
459
356
160
123
122
84
66
42
36
38
NuGel™ NRicher™ 6 (formerly NuGel
PROspector™) Protein Compression.
Neg.Cont.
53
76
35
24
3
19
0
0
2
0
0
A partial list of LC-MS/MS identification and spectral counts demonstrate
Imatinib interaction proteins from a common tissue homogenate, using
CCDP. Caffeine was employed as a non-specific control compound,
negative control was the final wash buffer.
PROfessor™ Product Sheet
Page 1
These non-reduced SDS-PAGE profiles show
that major high abundance bands are greatly
reduced. The low abundance bands are enriched
relative to their high abundance bands, visually
estimated to be 5-10X for most bands. Because
of its heterogeneity, the IgG band remains in
high abundance. The protein compression is
both tissue and species agnostic, with no bias
towards molecular weight or pI.
April 02, 2015
Product
NuGel™ NRicher™ Mx
(formerly NuGel
PROfessor™)
NuGel™ NRicher™ Mx
(formerly NuGel
PROfessor™)
# of
preps*
10
Item No.
Price
SRPRO-10
$450
50
SRPRO-50
$2025
The NuGel™ NRicher™ Mx (formerly NuGel PROfessor™) product kit includes all surface
reagents, binding and elution buffers and associated separations protocols. Each prep processes
approximately 0.5-1.0 mg total protein, and produces eluate sub-proteomes in 50 µl volumes in less
than 1 hour. Efficient protein compression may require higher loads, and will vary with sample type.
Kit Contains:
NuGel™ NRicher™ Mx
(formerly NuGel
PROfessor™) reagent
Cleanascite™ PRO reagent
suspension*
PRO-BB Binding Buffer, pH 6.0
PRO-WB Wash Buffer pH 7.0
PRO-EB Elution Buffer, pH 9.0
Spin-X microfuge filters
NuGel™ NRicher™ Mx
(formerly NuGel
PROfessor™) 10
150 mg
NuGel™ NRicher™ Mx
(formerly NuGel PROfessor™)
50
750 mg
0.5 ml
5 ml
3 ml
6 ml
0.5 ml
10
15 ml
30 ml
2.5 ml
50
*Refrigerate upon arrival.
Protocol
Step 1 - Sample Preparation: The protocol is based on 100 µl of tissue homogenates with a
soluble protein content in the 5 – 15 mg/ml range, per prep. Larger volumes of lower protein
content can also be used, with volumes proportionately adjusted throughout the protocol. For
applications where protein compression is desirable, higher protein loads will produce more protein
compression, for more details about this phenomena, please contact technical support. For
serum/plasma, we recommend at least 50 µl be applied for protein compression. It has not been
evaluated on membrane or insoluble protein content, but it is compatible with up to 0.1% Triton X100. The ideal pH for samples should be around 6-7.
Delipidation and removal of insoluble biomass is recommended. Cleanascite™ PRO supplied as part
of the kit, is a used for this purpose. Actual lipid concentration in biological samples can vary
greatly, so the ratios shown are only intended to provide general guidance in use.
1) Resuspend CleanasciteTM PRO by gentle shaking. It should be completely resuspended prior to
use.
2) In microfuge tube, add 1 volume of CleanasciteTM PRO to 5 volumes of the sample. Mix the
sample by gentle shaking for 10 minutes.
3) Centrifuge sample at 14,000 rcf (maximum microcentrifuge speed) for 3 minutes.
PROfessor™ Product Sheet
Page 2
April 02, 2015
4) Pipette out the “delipidated sample” into another tube. This will be the prepared sample for the
NuGel™ NRicher™ Mx (formerly NuGel PROfessor™) separations.
Step 2 - Surface Preparation. The NuGel™ NRicher™ Mx (formerly NuGel PROfessor™)
reagent is supplied in dry powder form. Weigh out 15 mg for each prep and place into the
Spin-X filters provided. Before using, tap each to ensure powders are at the bottom of the filter
cup.
1) Add 100 l of PRO-BB binding buffer to each reagent powder and mix for 3 minutes.
2) Centrifuge at [5,000-7,000]xg for 4 min. and discard the flow-through.
For Steps 3 & 4. All centrifugations are at [5,000-7,000]xg for 4 min.
Step 3 - Separations. All centrifugations are at [5,000-7,000]xg for 4 min.
1) Add 100 l of PRO-BB binding buffer and 100 l of “prepared sample” from above, to each
reagent powder (from Step 2). Mix until homogeneously resuspended, and shake the mixture for 10
minutes.
2) Add 250 l of PRO-WB wash buffer to each surface as a wash. Mix until homogeneously
resuspended, and shake the mixture for 10 minutes. Centrifuge and discard filtrate. Repeat wash
step 2 again. The bead is now enriched with non-specifically bound proteins. For LC-MS
sample preparation, an on-bead digestion protocol can be applied (protocol follows on
next page). Otherwise proceed to the next step.
Step 4 - Elution. Two options are available for elution:
A. For compound-centric displacement proteomic applications, OR
B. for protein compression, use full elution protocol.
A. For compound-centric displacement proteomic applications any challenging displacement
compound can be added into the PRO-WB wash buffer (subject to solubility constraints) and used
as an eluate. Use 50 l. Mix to homogeneously resuspend. Shake the sample for 10 minutes.
Centrifuge. Collect eluate filtrates for analyses.
B. To eluate the compressed proteins from the NuGel™ NRicher™ Mx (formerly NuGel
PROfessor™) surface, add 50 l of PRO-EB elution buffer to each surface. Mix to homogeneously
resuspend. Shake the sample for 10 minutes. Centrifuge. Collect filtrates as eluate fractions for
analyses. Elution buffer is pH 9.5, so any functional and/or activity measurements must compensate
for either higher pH, dilutions to neutrality, or buffer exchange.
Related Functional Proteomic Product - NuGel™ NRicher™ 6 (formerly NuGel
PROspector™)
Functional proteomics relies in part, on the functional or structural features of intact, non-denatured
proteins. While the terminology can often overlap, chemical and affinity-based proteomic profiles
can be considered a subset of functional proteomics. Both NuGel™ NRicher™ 6 (formerly NuGel
PROspector™) and NuGel™ NRicher™ Mx (formerly NuGel PROfessor™) support functional and
chemical proteomics and can:




Optimize drug compounds
Survey compound promiscuity
Deconvolute targets, elucidate mechanism of action
Identify phenotypic biomarkers
PROfessor™ Product Sheet
Page 3
April 02, 2015
Please consult our Functional & Chemical Proteomics Handbook online, to see how
NuGel™ NRicher™ Mx (formerly NuGel PROfessor™) and NuGel™ NRicher™ 6 (formerly
NuGel PROspector™) complement each other.
Suggested On-Bead Digestion Protocol
 After the final wash steps from step 7, add 50 µls of 10 mM DTT solution to the beads for
complete immersion, mix and incubate at 60°C for ½ hour.
 After cooling, add 50 µls of 50 mM iodoacetamide to the DTT/bead suspension, mix and
incubate in the dark for 1 hour.
 Centrifuge at 5000xg (medium setting, not max) for 3 mins, and discard supernatant.
 On-bead digestion is done by adding 100 µls of a 0.125 ug/uL (or calculated to a user
preferred ratio – typically 50-100:1 w:w, protein:trypsin) of MS-grade Trypsin to the
beads. Digest overnight at 37°C.
 Centrifuge at 5000xg (medium setting, not max) for 3 mins, and retain peptide filtrate.
 To further extract remaining peptides, add 100 µls of 10% solution of formic acid to the
beads.
 Incubate for 15 minutes at 37°C, centrifuge at 5000xg (medium setting, not max) for 3
mins, and add this volume to the first volume.
 Reduce to final volume using a SpeedVac.
PROfessor™ Product Sheet
Page 4
April 02, 2015
References
Matthew P. Kuruc, Swapan Roy. The Functional & Chemical Proteomics Handbook03/2014
Oka, Amita R., Matthew P. Kuruc, Ketan M. Gujarathi, and Swapan Roy. "Functional Proteomic
Profiling of Phosphodiesterases Using SeraFILE Separations Platform." International Journal of
Proteomics 2012 (2012).
New Chemical Proteomic Methods To Access Drug-Protein Interactions
US HUPO 2014. Frontiers in Proteomics: Advancing Biology through Technology and
Computation.
NuGel™ PROfessor™ abstract entitled "Compound-Centric Displacement Proteomics - An Advantaged
Method To Survey Small Molecule-Protein Interactions" poster board 096 presented at US HUPO
2014
CONTACT US
We welcome your questions and comments regarding our products.
Tel:
732-274-2866, 800-935-0628(North America) Mon – Fri 9am-6pm EST.
Fax
732-274-2899
Email
[email protected]
Mail
1 Deer Park Drive, Suite M, Monmouth Junction, NJ 08852
Web:
www.biotechsupportgroup.com
PROfessor™ Product Sheet
Page 5
April 02, 2015