Download Magic Lysis Buffer Improves the Efficiency of

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 B.
1Beth
1,
2
Breitkopf ;
Israel Deaconess Medical Center, Boston, 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 are necessary when probing in vivo tissue
sources, a focus of our laboratory. Many scientists have
attempted to optimize conditions for reducing nonspecific interactions and maximizing binding of true
binding partners with varying success. Since many
signaling complexes are bound through phosphorylation
events, we show that magic buffer preserves tyrosine
kinase activity in cell lysates.
The Buffer
The magic cell lysis buffer (ProNet LIVE! from ESI Source
Solutions, Woburn, MA) presented here is composed of a
specific molar ratio of a proprietary blend of a pluronic
non-ionic copolymer surfactant and a non-ionic detergent.
The buffer is supplied as a sterile filtered isotonic solution
at pH=7.6 in Tris-HCl. The buffer contains no actual magical
components.
Min
1
Yuan ;
2Harvard
John
3
Neveu ;
John M.
Medical School, Boston, MA;
Greater Sensitivity
80% less non-specific protein 150% increase in unique
binding
peptide coverage for
canonical hits
The Grb2 protein and its binding partners were immunoprecipitated from H929
multiple myeloma (MM) cell lysate in either magic buffer or 0.5% NP-40 buffer
and run by LC/MS/MS. Magic buffer showed more canonical binders and less
background (sticky) proteins.
3ESI
Source Solutions, Woburn, MA
Magic Buffer allows for the near complete capture of the
BCR/ABL complex from a p85 IP in K562 CML cells
Magic Buffer Shows Vast Improvement Over The
Leading Lysis Buffer for IP-MS in H929 MM Cells
Higher Specificity
1,
2
Asara
B)
A)
A). The IP-MS results from a p85 protein (PI3K) immunoprecipitation in K562 chronic myeloid
leukemia (CML) cell lysate from both buffer systems. Magic buffer identified more canonical
binding proteins and nearly captured the entire known complex. B). The BCR-ABL pathway
typical of K562 CML cells and H929 positive BCR-ABL MM cells.
Magic Buffer Helps Preserve Novel PI3K Interacting
Proteins in EGFR Driven Cancers
A)
Magic Buffer Captures More Canonical Grb2 Binders
Known to Be Important for Tyrosine Kinase Signaling
and Cell Proliferation in BCR-ABL Transformed Cells
B)
Methods
Sample Preparation: Cancer cell lines (H929, K562, A431)
were cultured and lysed using either magic buffer or 0.5%
NP-40 buffer with added phosphatase and protease
inhibitors to a final protein concentration of ~1-2 mg/mL.
The key nodal proteins Grb2, SHP2 or p85 (PI3K) involved
in growth signal transduction pathways were
immunoprecipitated (IP’d) using antibodies against the
endogenous proteins immunopurified for 2 hours at 4C
with magic or NP-40 buffer. Protein A beads were then
washed with the lysis buffer 3X and loaded onto SDS-PAGE.
Gels were stained with coomassie blue.
Mass Spectrometry: Short gel runs were excised above and
below the heavy IgG band at 55kD, digested with trypsin
overnight and run by LC/MS/MS with the Orbitrap Elite
(Thermo) couple to a EASY-nLCII (Thermo) at 300 nL/min
over a 60 min. gradient (DDA, Top 15, CID).
Proteins/peptides were identified using Mascot 2.4 against
the decoy Human database (UniProt) and quantified with
Scaffold 4 software (MS2 based spectral counting) at <1%
FDR.
Signaling Blots: We performed immunoblots for pTyr
proteins (pTyr-100 AB, Cell Signaling Tech) and PathScan
RTK Signaling Antibody Arrays (CST) to check for important
phospho-signaling proteins preserved in both the NP-40
and magic buffer systems.
A) The p85 IP from the EGFR mutated A431 cell line shows a novel interaction of p85 with
EPS-15 in addition to expected interactions (not shown). B) Other EGFR driven non-small
cell lung cancer (NSCLC) cells such as HCC827 also show p85-EPS15 binding and can be
verified with Western blots.
Grb2 was immunopurified from H929 cell lysate in both buffer systems and the
canonical binding partners were quantified via spectral counting after LC/MS/MS.
PathScan RTK Signaling Antibody Array Shows That Magic
Cell Lysis Buffer Can Preserve Phosphorylation Signals
Phosphotyrosine Blots of Cancer Cell IPs and Whole
Cell Lysates Show Higher Phosphorylation Intensity
with Magic Buffer
A)
B)
Whole Cell Lysate
Higher phosphorylation intensities across key signaling nodes using magic buffer vs. NP-40
Conclusion
A) Phosphotyrosine (pTyr) Western blots from p85 and Shp2 IPs using both magic
buffer and NP-40 buffer. B) PTyr blots from whole cell lysates of three different cancer
cell lines. Samples were normalized either to bait protein or actin levels.
• The magic cell lysis buffer (ProNet LIVE! ESI Source Solutions, Woburn, MA
www.esisourcesolutions.com) shows improved efficiency for IP-MS studies
(less background, more real interactions, etc.)
• Magic buffer better preserves phosphorylation activity
Acknowledgements
This work was partially supported by NIH grants 2P01CA120964 (J.M.A.) and S10OD010612 (J.M.A.)