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

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Oct 18, 2022 174 likes •286 views

Incorporation efficiency of heavy isotopic amino acids into OriGenes Mass Spec Standards Data prepared by Dr. Wei Yan Institute for Systems Biology Introduction OriGene manufactured the heavy isotope labeled MAP2K1 and MAPK1 by

SLIDE 1

Incorporation efficiency of heavy isotopic amino acids into OriGene’s Mass Spec Standards

Data prepared by Dr. Wei Yan Institute for Systems Biology

SLIDE 2

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
ver 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%.

SLIDE 3

The C13 and N15 labeled recombinant proteins as Mass-Spec internal standard

MAPK1 CTNNB1 MAP2K1 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.

SLIDE 4

Protein: MAP2K1 (Cat#: PH318460)

(Peptides detected by mass spec are highlighted in yellow)

Representative peptide: ISELGAGNGGVVFK (position: 71-84 mass: 1346.7194) Predicted m/z for +2 ion (M+2H)2+ : Light: 674.3670 Heavy: 678.3741

Peptides identified from MAP2K1 MS analysis

SLIDE 5

RT: 35.42 - 36.90 35.6 35.8 36.0 36.2 36.4 36.6 36.8 Time (min) 5000000 10000000 15000000 20000000 25000000 30000000 35000000 40000000 45000000 50000000 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 36.10 36.12 36.08 36.14 36.18 36.02 36.21 35.98 36.27 36.28 35.94 36.32 35.91 36.35 36.37 35.89 36.38 35.87 36.42 35.86 36.45 36.48 35.81 36.53 36.55 35.78 36.63 36.75 36.84 35.74 35.64 35.51 36.10 36.12 36.04 36.14 36.02 36.16 36.20 36.01 36.23 35.98 36.27 35.94 36.30 35.91 36.35 36.37 35.89 35.87 36.38 35.86 36.42 35.83 36.48 35.80 36.51 36.60 35.76 36.69 36.77 35.72 35.64 35.54 NL: 4.24E6 m/z= 674.36- 674.38 MS OR2010011 6_uk_12_pro tein_5_B NL: 5.28E7 m/z= 678.36- 678.38 MS OR2010011 6_uk_12_pro tein_5_B

HPLC spectra for co-eluted light and heavy peptides of tryptic digested MAP2K1

Light Heavy

Calculated ratio (L/H) by the area under curve (AUC): 0.08

SLIDE 6

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] 674 675 676 677 678 679 680 681 682 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 678.3734 678.8714 679.3708 679.8716 674.8678 674.3674 677.8745 675.3689 680.3732 680.8758 677.4496 676.4451 675.8715 681.8777 673.9529

Heavy peptide Light peptide

MS spectra of representative light and heavy peptides of tryptic digested MAP2K1

SLIDE 7

Protein: MAPK1 (Cat#: PH304703)

(Peptides detected by mass spec are highlighted in yellow)

Representative peptide: GQVFDVGPR (position: 16-24 mass: 973.4981) Predicted m/z for +2 ion (M+2H)2+ : Light: 487.7563 Heavy: 492.7605

Peptides identified from MAPK1 MS analysis

SLIDE 8

RT: 28.73 - 30.35 28.8 29.0 29.2 29.4 29.6 29.8 30.0 30.2 Time (min) 5000000 10000000 15000000 20000000 25000000 30000000 35000000 40000000 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 29.45 29.42 29.46 29.52 29.35 29.55 29.33 29.61 29.32 29.65 29.31 29.68 29.72 29.28 29.77 29.79 29.26 29.84 29.88 29.24 29.93 29.98 29.22 30.08 30.14 29.17 29.11 29.43 29.42 29.47 29.39 29.35 29.51 29.55 29.33 29.56 29.32 29.57 29.65 29.31 29.66 29.28 29.68 29.72 29.27 29.77 29.84 29.88 29.24 29.93 29.98 29.22 30.08 29.19 30.17 29.13 28.81 29.04 NL: 4.51E6 m/z= 487.75-487.77 F: FTMS + p NSI Full ms [300.00-1800.00] MS OR20100116_uk_14_pr

tein_6_B

NL: 4.17E7 m/z= 492.75-492.77 F: FTMS + p NSI Full ms [300.00-1800.00] MS OR20100116_uk_14_pr

tein_6_B

Light Heavy

Calculated ratio (L/H) by the area under curve (AUC): 0.11

HPLC spectra for co-eluted light and heavy peptides of tryptic digested MAPK1

SLIDE 9

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] 487 488 489 490 491 492 493 494 495 496 497 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 492.76 493.26 495.76 493.76 487.76 496.27 492.26 488.26 494.26 495.26 496.77 488.76 490.76 494.76 491.57 492.07 497.27 489.26 487.28 490.21