Transcript ASMS 2013-2 - ESI Source Solutions

Magic Lysis Buffer Improves the Efficiency of Immunoprecipitation-LC/MS/MS (IP-MS) with
Less Non-Specific Interactions and Stronger Retention of Binding Protein Partners
Susanne
1Beth
1
Breitkopf ;
Min
Israel Deaconess Medical Center, Boston, MA;
1
Yuan ;
2Harvard
John
3
Neveu ;
John M
Medical School, Boston, MA;
1,
2
Asara
3ESI
Source Solutions, Woburn, MA
Introduction
Immunoprecipitation (IP) - Tandem Mass Spectrometry
(IP-LC/MS/MS) has long suffered from contamination
with non-specific protein interactions that suppress
true bait-prey binding partners. In addition, caution
must be taken about the stringency of lysis buffer since
it can strip the bait protein of true binding partners.
These problems are especially true for IPs using
antibodies against endogenous proteins. However, IPs
using endogenous antibodies is necessary when
probing in vivo tissue sources, a focus of our
laboratory. Many scientists have attempted to optimize
conditions for reducing non-specific interactions and
maximizing binding of true binding partners with
varying success. The magic buffer presented here
composed of a specific molar ratio of a pluronic nonionic copolymer surfactant and a non-ionic detergent
accomplishes that goal.
Experimental:
The magic buffer was used as a direct replacement for
alternative detergents in common cell lysis and
immunoprecipitation procedures (IP) for comparison.
The buffer, which actually contains no magical
components, extracts even membrane proteins with
high efficiency, retains full native protein activity,
allowing the exceptional specificity in IP results shown
here. This improved native activity can be
Magic Buffer shows vast improvement over the
leading lysis buffer for IP-MS studies:
Higher Specificity
Greater Sensitivity
Magic Buffer allows for the near complete capture of
the BCR/ABL complex from a p85 IP in K562 chromic
myeloid leukemia cells:
80% less non-specific
protein binding
150% increase in unique
peptide coverage for
canonical hits
Magic Buffer May Help Preserve
Novel PI3K Interacting Proteins
Magic buffer captures more canonical Grb2
binders known to be important for tyrosine
kinase signaling and cell proliferation in
BCR/ABL transformed H929 cells:
PathScan RTK Signaling Antibody Arrays, Cell Signaling Tech.
Show That Magic Buffer Can Preserve Phosphorylation
Signals over the Common Lysis Buffer Systems
Methods
Cancer cell lines were cultured and lysed using either
magic buffer or 0.5% NP-40 buffer. Solid human tumor
samples were also lysed in either magic buffer or 0.5%
NP-40. The key nodal proteins Grb2 or p85 (PI3K)
involved in signal transduction pathways were
immunoprecipitated using antibodies against the
endogenous proteins. Protein complexes were
immunopurified overnight with magic or NP-40 buffer.
IPs were then washed and loaded onto SDS-PAGE.
Short gel runs were excised below and above 55kD,
digested with trypsin and run by nLC/MS/MS with the
Orbitrap Elite. Proteins/peptides were identified using
Sequest and quantified with Scaffold (MS2 based
spectral counting) or used MaxQuant for both
identification and MS1 Label-Free Quantification. We
performed immunoblots for pTyr and related signaling
proteins.
H929 Multiple Myeloma Cells Grb2 IP
Phosphotyrosine Blots of Cancer Cell
Immunoprecipitations (IPs) and Whole
Lysates Show Higher Phosphorylation
Intensity with Magic Buffer