Analysis of the human phosphatase system PP2A Application Note 1: CaptiVate™ technology Summary PP2A subunits PPP2CB, PPP2R1A and PPP2R2A The list of protein ID`s were filtered against a back- were stably integrated in HEK293 cells by the FRT ground database generated in-house (Flippase recombination target) system A protein network was assembled and proteins were SH-double affinity purification was performed grouped into classes relevant to PP2A function Samples were analysed by LC-MS/MS using an LTQ- The application of CaptiVate™ technology revealed Orbitrap mass spectrometer new interactors of the PP2A system Introduction The serine/threonine phosphatase PP2A is a highly con- B-type regulatory subunits (>15) and thereby exceeds the served phosphatase complex with important biological func- number of catalytic and scaffolding subunits (n=2) within the tion and has been linked to regulate a variety of vital cellular human system. This in turn provides a molecular basis for the processes including apoptosis, transcription and cell prolif- assembly of PP2A into a multitude of possible heterotrimeric eration and cellular transformation [1]. phophatase complexes. It is assumed that the B-type regu- latory subunits provide substrate binding and specificity to The majority of PP2A complexes exist as a heterotrimeric as- individual heterotrimers and thereby allowing the control of sembly between a catalytic, a scaffolding and a regulatory diverse biological processes by combinatorial assembly of B subunit. The human genome encodes a large number of the phosphatase complex. Methods — CaptiVate™ technology Cell line generation Double affinity purification nano-LC-Orbitrap-MS Data filtering Generation of isogenic cell Highly efficient Strep/HA- Sensitive protein identifica- Filtering vs. background data- lines expressing the bait pro- double affinity purification tion using nano-LC-Orbi- base to get specific interactors tein using the FRT system trap-MS Results Here, we present the analysis of PP2A phosphatase specific Cell lines expressing the PP2A subunits PPP2RCB (cata- subunits to monitor the performance and applicability of the lytic subunit), PPP2R1A (scaffolding subunit) and PPP2R2A CaptiVate™ technology. First, we monitored the efficiency (regulatory B subunit) were used to generate a phosphatase of the Strep/HA-double affinity purification by using Western interaction network of PP2A. Following SH-purification and blotting (Figure 1). We routinely obtain a purification yield of LC-MS/MS analysis using an LTQ-Orbitrap mass spectro- 30-40% of the starting material, which is among the highest meter the list of protein ID`s was filtered against a back- yields reported for double affinity purification protocols [2]. ground database generated in-house. From the resulting list of proteins a protein interaction net- First step — Second step — work was assembled and the protein interaction database Strep anti-HA Biogrid was used as a reference database to detect new pro- L SN1 E1 SN2 E2 WB: tein interactions (Figure 2). SH-PPP2R2A a-HA In total we identified 69 proteins, which correspond to 103 protein interactions associated with the bait proteins. Among 0.3 0.3 1.5 1.5 1.5 % input the 103 interactions, 36 interactions were previously known according to Biogrid information (Figure 2, blue lines), where- Figure 1: Monitoring of SH-double affinity purification. as 67 protein interactions did not occur in the database (Fig- Cells expressing the SH-tagged version of PPP2R2A were lysed. ure 2, red lines). In order to annotate the identified proteins, The lysate (L) was applied to StrepTactin beads and eluted with lysis GO annotations were used and proteins were grouped into buffer containing biotin (E1). The second purification step was per- classes relevant to PP2A function (cell signalling, apoptosis, formed using anti-HA agarose. The final eluate was obtained by pH cell proliferation, protein folding, transcriptional control). shift elution (E2). SN1: supernatant after first purification; SN2: su- pernatant after second purification. PPP2R2B CCT3 CCT8 PPP2R2D CCT6A TCP1 PPP2R5E R3HCC1 CCT2 CCT4 PPP2R3A BAT2D1 CCT7 CCT5 PPP2R3B WIZ PPP2R5A WDSOF1 TPX2 Bait PPP2R5C FAM122A GTSE1 STRN3 PPP2R5D MAP7D2 SIK2 Prey STRN MAP7 FOXC1 STRN4 CDCA4 CRTC3 CCDC9 BAG2 catalytic subunit PPME1 PPP2R2A regulatory B subunit scaffolding subunit PPP2CA PPP2R1A SEMG2 RBM7 protein PPP2CB PPP2R1B SERTAD4 ATXN2L methylesterase ZCCHC8 apoptosis IGBP1 PPP4C SRP72 IQGAP1 MOBKL3 INTS12 cell proliferation HP INTS1 PREI3 KIF22 MYL6 CDCA2 LPP FGFR1OP protein folding SRP14 SGOL1 CTTNBP2NL SEMG1 LIMD1 signal transduction C20orf117 FAM40A PRR14 KIAA0692 TBCCD1 PPFIA1 C22orf30 DSP regulation of transcription Figure 2: PP2A interaction network. Protein-protein interactions and different classes of protein complexes within the human PP2A interaction network are shown. Blue lines indi- cate previously known interactions; red lines indicate interactions not occurring in the Biogrid database. V 02/2010 Dualsystems Biotech AG Dualsystems Biotech Inc. 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