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 Breitkopf1; Min Yuan1; John Neveu3; John M Asara1, 2 1Beth Israel Deaconess Medical Center, Boston, MA; 2Harvard Medical School, Boston, MA; 3ESI Source Solutions, Woburn, MA

IntroductionImmunoprecipitation (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 non-ionic copolymer surfactant and a non-ionic detergent accomplishes that goal.

MethodsCancer 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.

Magic Buffer shows vast improvement over the leading lysis buffer for IP-MS studies:

Greater Sensitivity

150% increase in unique peptide coverage for

canonical hits

Higher Specificity

80% less non-specific protein binding

Magic Buffer allows for the near complete capture of the BCR/ABL complex from a p85 IP in K562 chromic

myeloid leukemia cells:

Magic buffer captures more canonical Grb2 binders known to be important for tyrosine

kinase signaling and cell proliferation in BCR/ABL transformed H929 cells:

H929 Multiple Myeloma Cells Grb2 IP

Magic Buffer May Help Preserve Novel PI3K Interacting Proteins

Phosphotyrosine Blots of Cancer Cell Immunoprecipitations (IPs) and Whole Lysates Show Higher Phosphorylation

Intensity with Magic Buffer

PathScan RTK Signaling Antibody Arrays, Cell Signaling Tech. Show That Magic Buffer Can Preserve Phosphorylation

Signals over the Common Lysis Buffer Systems

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