US 20060019285A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0019285 A1 Horecka et al. (43) Pub. Date: Jan. 26, 2006 (54) ANALYSIS OF INTRACELLULAR Publication Classification MODIFICATIONS (51) Int. Cl. CI2O I/68 (2006.01) (76) Inventors: Joseph Horecka, Fremont, CA (US); GOIN 33/53 (2006.01) Peter Fung, Sunnyvale, CA (US); (52) U.S. Cl. .................................................. 435/6; 435/7.1 Richard M. Eglen, Los Altos, CA (US) (57) ABSTRACT Correspondence Address: Improved methods of determining the intracellular State of a PETERS VERNY JONES & SCHMITT, L.L.P. protein as well as modifications of the protein are provided 425 SHERMANAVENUE by introducing a Surrogate fusion protein comprising a SUTE 230 member of an enzyme fragment complementation complex and a target protein. After exposing cells transformed with PALO ALTO, CA 94.306 (US) the Surrogate fusion protein to a change in environment, e.g. Appl. No.: 11/170,123 a candidate drug, the cells are lysed, the lysate Separated into (21) fractions or bands, conveniently by gel electrophoresis and (22) Filed: Jun. 29, 2005 transferring the proteins by Western blot to a membrane. The bands on the membrane are developed using the other Related U.S. Application Data member of the enzyme fragment complementation complex and a Substrate providing a detectable Signal. The method is (60) Provisional application No. 60/584,709, filed on Jun. found to provide high sensitivity and the ability to observe 30, 2004. modifications of the target protein. Patent Application Publication Jan. 26, 2006 Sheet 1 of 7 US 2006/0019285 A1 FIG. 1 Patent Application Publication Jan. 26, 2006 Sheet 2 of 7 US 2006/0019285 A1 100 - 52 80 2. 53 60 & 40 in N 20 SS O 0 2 4 8 16 32 TNF-O TREATMENT TIME (min) F/G. 2 TNF-C (min) 0 2 4 8 16 32 IkB-PL Patent Application Publication Jan. 26, 2006 Sheet 3 of 7 US 2006/0019285 A1 Patent Application Publication Jan. 26, 2006 Sheet 4 of 7 US 2006/0019285 A1 Q 2N c) d Oh (19 - Z St - O - - 5 o O O C on3 CA- 2. aC 21 1. CX 3 s d N v- d 9 - St. t < 3 2. H 92 f S - O v v O s UC) S |OJuOO O O % Patent Application Publication Jan. 26, 2006 Sheet 5 of 7 US 2006/0019285 A1 8/ G/|!LJ|||- Z8 |OZZ 00|| 9Ç0 09 #7 -08 |OJuO3 O JO % |-0 Patent Application Publication Jan. 26, 2006 Sheet 6 of 7 US 2006/0019285 A1 loodWNA'sgºlfiunsel-ºgigZ-GIJA 8£##7 |-- 79 |6 00|| ZOI! 09 0 |OJuOO9/08 O JO % *07 Patent Application Publication Jan. 26, 2006 Sheet 7 of 7 US 2006/0019285 A1 a. 2 CC 1. 2 s 4. (M) d 2 C O o N Z :::::s45:x: ts 3. NŽ 2 5 Si g Z g N 5 s : SR 3 Z2222222222 N 75 g. e S 5 S (M) N S Z - OO s N to s = S Z2 i N N O C O O C 3 OO CO r cN Oulu.OO O JO % US 2006/0019285 A1 Jan. 26, 2006 ANALYSIS OF INTRACELLULAR compound as a drug. Proteins can undergo numerous modi MODIFICATIONS fications, Such as partial or total degradation, glycosylation, ubiquitination, phosphorylation or dephosphorylation, CROSS-REFERENCE TO RELATED acetylation, acylation, prenylation, etc. Being able to discern APPLICATIONS the existence of different aspects of the same protein is important in understanding pathways, Such as identifying 0001. This application claims priority from U.S. Provi the major and minor Status of the parent protein and the sional Patent Application No. 60/584,709 filed on Jun. 30, modified protein, where the effect of a candidate compound 2004, which is hereby incorporated by reference in its can be analyzed. Also, by being able to observe patterns of entirety. variations in the protein analogs and partial degradation products, one can identify cellular pathways and their inter REFERENCE TO SEQUENCE LISTING action with other proteins. 0002 Applicants assert that the paper copy of the 0010. There is, therefore, Substantial interest in develop Sequence Listing is identical to the Sequence Listing in ing techniques that will allow the efficient investigation of computer readable form found on the accompanying com cellular proteins, the effect of changes in the cellular envi puter disk. Applicants incorporate the contents of the ronment on the protein, and being able to detect low levels Sequence listing by reference in its entirety. of a protein of interest in the presence of large amounts of BACKGROUND OF THE INVENTION other proteins that may have analogous migration aptitudes. Relevant Literature 0003) 1. Technical Field 0011 U.S. Patent Nos. 6,680,208 and 6,677,128 and 0004. The field of this invention is proteomic assays. WO94/29725 describe various Western blotting techniques. 0005 2. Background PCT/US02/27497 describes the use of fusion proteins employing enzyme fragmentation complexes for determin 0006 AS more is learned about cellular pathways, the interactions of microorganisms with mammals, the growth ing the Status of the fusion protein in a cell. and development of organisms, both prokaryotic and SUMMARY OF THE INVENTION eukaryotic, there is an increasing need for improved meth ods of identifying protein members involved with these 0012. The fate of cellular proteins is determined by various processes. Also, as proteins and their function are employing a genetically modified cell expressing one or identified, there is an increasing interest in being able to more proteins of interest fused to a fragment that is a modulate the activity of proteins associated with diseased member of an enzyme fragment complementation (“EFC) States. By Screening for active compounds that modulate the pair. The lysate of the cell is separated, conveniently activity, one is interested, not only in identifying the effect employing Western blotting, and the fractions or bands of the compound on the target protein, but also any effects developed using the other member of the EFC pair and a the compound may have on other proteins present in the cell Substrate. The observed fractions or bands are limited to or medium. those proteins that comprise the fused fragment, allowing for detection of any form of the protein(s) of interest that 0007. There are numerous ways to separate components retains the fragment. of a complex mixture based on differences in the compo nents of mass, mass-to-charge, conformation, etc. Gel elec trophoresis has been a major methodology for analyzing BRIEF DESCRIPTION OF THE FIGURES mixtures of proteins. Native proteins and denatured proteins 0013 FIG. 1 shows the detection on blots of B-arrestin2 are readily Separated by their migration rate, which for the fusion proteins tagged with both ProLabel and the c-myc most part can be related to their molecular weight. Numer epitope. EAStern blots (upper panels) were photographed ous techniques have been developed for identifying the under white light after incubation with the chromogenic separated protein bands. The Western blot was developed to Substrate X-Gal for the indicated times. 1A is an EAStern identify proteins. As part of the Western blot, the proteins are Blot with overnight incubation; 1B is an EAStern Blot with Separated by electrophoresis in a gel, followed by transfer of 2 day incubation; The Western blots (lower panels) were the protein bands to a membrane. The bands are then developed with the chemiluminescent substrate CDP-Star developed by various techniques, Such as labeled antibodies and detected with a CCD camera using the indicated expo or a non-specific protein dye. Other methods of Separation Sure times. Lane 1, prestained MW marker (Invitrogen, Cat. include chromatography, e.g. HPLC, FPLC, capillary elec No. LC5925). Lanes 2-4, lysates of CHO-K1 cells tran trophoresis, etc. siently transfected with plasmids pPL-myc-barr2, pbarr2 0008. The Western blot methodology has many desirable myc-PL, and pEGFP-C1, respectively; 1 C is a Western Blot, properties for identifying proteins in a mixture. However, PAb, 6 min incubation; 1D is a Western Blot, 12 minutes even with 2D protocols, where one is dealing with a lysate incubation; or comparable mixture, there can be protein overlap, diffi 0014 FIG. 2 is a graph showing the amount of IKf8-PL culties in detecting low amounts of a component, inadequate protein present in lysates after HeLa IKf8-PL stable cells Separations, inability to detect modifications of the protein were treated with TNF-C. for the indicated time periods. EFC and the like. values are expressed as percentages of untreated control 0009 Today, one is interested in determining many cells (“0” time point); aspects of the life of a protein in a cell, particularly in 0015 FIG.3 shows EAstern blot detection (upper panel, response to a change in the environment, e.g. a candidate A) of IKf8-PL using the chemiluminescent substrate Beta US 2006/0019285 A1 Jan. 26, 2006 Glo and a 3 min CCD camera exposure. Molecular weight 0024. Because of the convenience of gel electrophoresis marker sizes (kD) are indicated on the left. The anti-actin and Western blotting in allowing one to view the various Western blot (lower panel, B) serves as a loading control. fractions Simultaneously and compare their intensity, West Samples are the same as those described in FIG. 2; ern blotting is the preferred detection System. The membrane of the Western blot permits the simultaneous addition of the 0016 FIG. 4 shows increased CCD camera exposure necessary reagents to the fusion protein and its fragments, So times (in minutes, lower-right corner of each panel, 4A, 5 as to have a comparable concentration of the added reagents.