isotopic amino acids into OriGenes Mass Spec Standards Data prepared - PowerPoint PPT Presentation

Incorporation efficiency of heavy isotopic amino acids into OriGene’s Mass Spec Standards Data prepared by Dr. Wei Yan Institute for Systems Biology

Introduction • OriGene manufactured the heavy isotope labeled MAP2K1 and MAPK1 by transfecting HEK293T cells with over-expression clones. • The cells were cultured in heavy isotope medium for over 10 generations and the proteins were isolated using anti-DDK affinity column. • The heavy labeled proteins were sent to ISB for MS analysis and the incorporation efficiency were determined. • For both samples, the incorporation efficiency is over 90%.

The C13 and N15 labeled recombinant proteins as Mass-Spec internal standard MAP2K1 CTNNB1 MAPK1 MAPK3 188 98 62 49 38 28 17 14 MAP2K1 and MAPK1 were chosen for Mass Spec analysis to determine heavy amino acid incorporation efficiency.

Peptides identified from MAP2K1 MS analysis (Peptides detected by mass spec are highlighted in yellow) Protein: MAP2K1 (Cat#: PH318460 ) Representative peptide: ISELGAGNGGVVFK (position: 71-84 mass: 1346.7194) Predicted m/z for +2 ion (M+2H) 2+ : Light: 674.3670 Heavy: 678.3741

HPLC spectra for co-eluted light and heavy peptides of tryptic digested MAP2K1 RT: 35.42 - 36.90 36.10 36.12 NL: 36.08 4.24E6 36.14 4000000 m/z= 36.18 36.02 674.36- 3500000 674.38 MS 36.21 OR2010011 35.98 36.27 6_uk_12_pro 3000000 tein_5_B 36.28 35.94 2500000 36.32 35.91 Light 2000000 36.35 36.37 35.89 1500000 36.38 35.87 36.42 35.86 1000000 36.45 36.48 35.81 36.53 36.55 500000 35.78 36.63 36.75 36.84 35.74 35.51 35.64 0 36.10 36.12 NL: 5.28E7 36.04 36.14 50000000 36.02 m/z= 36.16 678.36- 45000000 36.20 36.01 678.38 MS 36.23 OR2010011 40000000 35.98 6_uk_12_pro 36.27 35000000 tein_5_B 35.94 36.30 30000000 35.91 25000000 36.35 36.37 35.89 20000000 35.87 36.38 Heavy 15000000 35.86 36.42 35.83 10000000 36.48 35.80 36.51 5000000 36.60 35.76 36.69 36.77 35.72 35.54 35.64 0 35.6 35.8 36.0 36.2 36.4 36.6 36.8 Time (min) Calculated ratio (L/H) by the area under curve (AUC): 0.08

MS spectra of representative light and heavy peptides of tryptic digested MAP2K1 OR20100116_uk_12_protein_5_B #3350 RT: 36.10 AV: 1 NL: 5.14E7 T: FTMS + p NSI Full ms [300.00-1800.00] 678.3734 100 95 90 678.8714 85 80 75 70 Heavy peptide 65 60 55 50 45 679.3708 40 35 30 Light peptide 25 20 679.8716 15 10 674.8678 674.3674 677.8745 675.3689 680.3732 5 680.8758 676.4451 677.4496 675.8715 673.9529 681.8777 0 674 675 676 677 678 679 680 681 682 m/z

Peptides identified from MAPK1 MS analysis (Peptides detected by mass spec are highlighted in yellow) Protein: MAPK1 (Cat#: PH304703 ) Representative peptide: GQVFDVGPR (position: 16-24 mass: 973.4981) Predicted m/z for +2 ion (M+2H) 2+ : Light: 487.7563 Heavy: 492.7605

HPLC spectra for co-eluted light and heavy peptides of tryptic digested MAPK1 RT: 28.73 - 30.35 29.45 NL: 4.51E6 29.42 4500000 29.46 m/z= 487.75-487.77 F: FTMS + p NSI Full ms 29.52 4000000 29.35 29.55 [300.00-1800.00] MS OR20100116_uk_14_pr 3500000 otein_6_B 29.33 29.61 3000000 29.32 29.65 2500000 29.31 29.68 2000000 29.72 29.28 29.77 Light 1500000 29.26 29.79 29.84 29.88 1000000 29.24 29.93 29.98 29.22 500000 30.08 30.14 29.17 29.11 0 29.43 NL: 4.17E7 29.42 29.47 m/z= 492.75-492.77 F: 29.39 40000000 29.35 FTMS + p NSI Full ms 29.51 [300.00-1800.00] MS 29.55 35000000 OR20100116_uk_14_pr 29.33 otein_6_B 29.56 30000000 29.32 29.57 25000000 29.65 29.31 29.66 29.28 20000000 29.68 29.72 29.27 15000000 29.77 29.84 10000000 29.88 29.24 Heavy 29.93 29.98 29.22 5000000 30.08 29.19 30.17 29.13 28.81 29.04 0 28.8 29.0 29.2 29.4 29.6 29.8 30.0 30.2 Time (min) Calculated ratio (L/H) by the area under curve (AUC): 0.11

MS spectra of representative light and heavy peptides of tryptic digested MAPK1 OR20100116_uk_14_protein_6_B #2428 RT: 29.43 AV: 1 NL: 4.15E7 F: FTMS + p NSI Full ms [300.00-1800.00] 492.76 100 95 90 85 80 75 Heavy peptide 70 65 493.26 60 55 50 45 40 35 30 25 Light peptide 495.76 493.76 20 15 487.76 496.27 492.26 10 488.26 494.26 495.26 5 492.07 496.77 488.76 490.76 494.76 491.57 487.28 489.26 490.21 497.27 0 487 488 489 490 491 492 493 494 495 496 497 m/z

Summary • OriGene C13 and N15 labeled MS protein standards exhibit high purity. • High percentage of peptide coverage was observed by MS analysis. • Both the HPLC and MS spectra demonstrated that the overall incorporation efficiency of heavy labeled isotopic amino acids for OriGene heavy labeled Mass Spec protein standards is over 90%.