LC-MRM Quantification of Src-Family Kinases using Protein Standards Expressed in Cell-Free Systems Elizabeth Remily-Wood,1 Michael Rosenblatt,2 Robin Hurst,2 Fumi Kinose,1 Eric Haura,1 Mark Meads,1 William Dalton,1 Kenneth Shain,1 John Koomen1 PROTEOMICS 1H. Lee Moffitt Cancer Center, Tampa, FL; 2Promega, Madison, WI

Overview Results for Protein Synthesis and Purification Figure 4 : Stable-Isotope Label Incorporation in Protein Standards. Table 2: Average Stable- Transition ratios were compared for light (A) and stable isotope-labeled Isotope Incorporation in ƒHigh sequence homology associated with SRC family of tyrosine Figure 2: Capture Efficiency during Purification of SRC Family Kinases (SFKs) protein standards for both heavy (B) and light forms (C) of each Expressed Protein Standards. kinases (SFK) using Magnetic HaloTag® Resin. SM = Starting Material FT = Flowthrough peptide. Isotope incorporation (%) was determined by ratio of heavy ƒPrior attempts to detect unique peptides unsuccessful % Isotope (Green) to light (Red) signals (D). Values for individual peptides Protein ƒ Limited knowledge of SFK expression in cancer cell lines Incorporation ranged from 100% to 91.5% ƒ Lack of access to specific antibodies 75 kDa 75 kDa SRC 98.1 LCK: LIEDNEYTAR y y y y ƒProteins expressed (both light and heavy) in a cell-free model, then 8 7 6 4 FYN 98.4 purified for analysis 25 kDa 25 kDa YES1 98.4 ƒExpressed proteins act as stable isotope-labeled standards (SIS) for A B C D SM FT SM FT SM FT 14.0 11.3 11.3 11.3 LCK 98.5 both relative and absolute quantification in cell line models SM FT SM FT SM FT 1.2 2.0 6) ƒ Quantify all endogenous peptides using one standard LCK FGR FER LYN YES1 BLK 200 6) HCK 98.6 ) 65 80 72 22 57 64 3 20 0.8 LYN 98.6 x 10 ity (10^ ity y ity Binding Efficiency (%) Binding Efficiency (%) (10^ ity 1.0 BLK 98.6

y ( ity 100 Introduction 10 Intens Intens (10^3) Intens 0.4 FGR 98.8

Methods for LC-MRM Analysis Intens The SRC family of tyrosine kinases includes Yes1 FRK 98.8 11.3 members with high protein sequence homology (Src, Src 0 0.0 0 0.0 SRMS 99.6 Lyn Fyn SFK proteins were denatured in 8 M Urea/100 mM AB, reduced, alkylated, and digested 12.5 15.5 11.0 12.0 11.0 12.0 11.0 12.0 Fyn, Yes, Lck, Hck, Lyn, Blk, Fgr, Frk, and SRMS) FER 98.1 Fgr with trypsin, then ziptipped before LC-MRM analysis. Peptides were selected with the Retention Time Retention Time Retention Time Retention Time (Fig. 1). Signaling mediated by these proteins Hck control cell proliferation and migration; thus, they Lck following parameters: 7-24 amino acids in length, no Methionine or Cysteine residues, Figure 5: Detection of SFKs in lung cancer lysate and Confirmation using Expressed SIS Proteins. play major roles in cancer development and Blk and no NxT or NxS sequences. All y-ions were screened from y3 to y(n-1). All samples Frk were run on a TSQ Quantum Ultra (Thermo) and data were analyzed with Skyline 1.1 HCC 827 cells were lysed and then spiked with a mix of heavy labeled SFK proteins. The mixture was progression. Detailed examination of SRC family Srms kinases (SFKs) can elucidate cancer biology, and (Maclean et al. Bioinformatics 2010, 26, 966.). digested overnight with trypsin, ziptipped and screened by LC-MRM for both light and heavy peptides. Brk peptide-based quantitative assays for the expression The ratio of light to heavy peptides from the cell lysate was calculated (A) and compared to the ratio of a of each SFK can have utility in deciphering which of Fer Results for LC-MRM Analysis sample containing only the SIS expressed protein mixture (B). Detection was confirmed from the 17.20 17.63 18.06 18.16 the family members is expressed in tumor cells. increased L/H ratio. LIEDNEYTAR is used as example. Detection of unique peptides for selected SFKs Table 1: Peptide Candidates for Assay Development. For each SFK, the number of in digests of HCC827 whole cell lysate (C). Figure 1: Section of unique peptides is listed; then, peptides found in more than one protein. Candidates were LC-MRM assays can quantify the expression of each dendrogram showing A BLK: verified by matching the retention times and fragmentation patterns of both light and 600 B 100 C SFK using unique peptides from each sequence. sequence similarity 3) 19.0 SRC: EGYVPSNFVAR Applied in parallel, all SFK proteins can be detected between protein heavy standard proteins individually and mixed together. 3 19.0 GPSAAFAPAAAEPK 9.46E3 kinase domains 8.11E3 and quantified in a single experiment, providing Peptide Proteins Unique (10^ ity FER: (adapted from Protein 400 6) additional utility over immunoblotting. Expressed Peptides 2 LQDWELR Manning et al. EVLDQVER SRC, FYN, YES1 2.81E4 proteins aid assay development and serve as stable Intens Science 2002, 298, GAYSLSIR FYN, YES1, FGR SRC 5 (10^ ity isotope-labeled standards (SIS). LYN: FYN: 1912-1934). FYN 3 200 50 GSLLDFLK HCK, SRC 1 SLDNGGYYISPR QLLSFGNPR Intens 19.0 ve Abundance 1.09E4 Methods for Protein Synthesis and Purification YES1 2 ti 6.89E3 IADFGLAR LCK, FYN, YES1, LYN, 19.0 ƒSFKs acquired from Promega (through Find My Gene™) in pF1K T7 HCK, FGR, BLK LCK 5 0 0 Rela Flexi® Vector then transferred to the pFN19A HaloTag® T7 SP Flexi HCK 6 18 19 20 18 19 20 LDNGGYYITTR FYN, YES1 Retention Time Retention Time Vector LIEDNEYTAR LCK, FYN, YES1, SRC LYN 5 Light to Heavy Ratio ƒLight proteins were expressed with TnT® SP6 High-Yield Wheat Germ VADFGLAR SRC, FRK, SRMS BLK 8 0 0.1124 0.0265 14 15 16 17 18 19 20 Protein Expression System VIEDNEYTAR LYN, HCK FGR 6 Time (min) ƒHeavy proteins were expressed with TnT® SP6 High-Yield Wheat Germ FRK 5 Conclusions Protein Expression System (-Amino Acids) with the addition of L-Arginine Figure 3: Selected SFK Unique SRMS 4 (U-13C ; U-15N ) and L-Lysine (U-13C ; U-15N ) Peptides from LC-MRM Screens. 6 4 6 2 FER 10 • All screened SFK proteins produced > 2 unique peptides for LC-MRM assay development. ƒ The DNA concentration for both heavy and light proteins was 0.16 µg/µl LYN: GSFSLSVR BLK: EGYVPSNFVAR FGR: SSITLER YES: LLLNPGNQR • Stable isotope incorporation for expressed protein standards > 98%. of reaction and incubated at 25°C for 90 minutes with shaking (1400 rpm) 1.2 15.1 12.3 • In protein mixtures, unique SFK peptides were able to be identified with LC-MRM ) ) ) ) 14.8 6 3 6 6 14.3 2.0 400 2.0 • Endogenous peptides for SFK proteins identified in digests of whole cell lysates. ƒ Src, Hck, Lyn, YES1, BLK, LCK and FER were purified using Magne™ x 10 x 10 x 10 x 10 0.8 HaloTag®Beads (G7281, Promega) SRMS, FRK, FGR and FYN were y ( ity ( ity y ( ity y ( ity Acknowledgments 1.0 200 1.0 purified as HaloTag® fusions 0.4 Moffitt Proteomics is supported by the US Army Medical Research and Materiel Command under Award No. Intens Intens Intens ƒExpressed protein concentration ranged from 5-10 µg/ml Intens 0.0 0.0 0 0.0 DAMD17-02-2-0051, the National Cancer Institute under Award No. P30-CA076292, and the Moffitt Foundation. 13.5 14.5 15.5 14 15 16 14 16 11.5 12.5 Triple quadrupole mass spectrometers were purchased with grants from the Bankhead-Coley Cancer Research Retention Time Retention Time Retention Time Retention Time program of the Florida Dept. of Health (06BS-02-9614, 09BE-04).