Genomic Health Evaluation of Genomic Health Evaluation of Corona Charged Aerosol Detection Corona Charged Aerosol Detection Comparing Tryptic Digest with UV and CAD Chris Crafts Chris Crafts 06/22/11
Sample Preparation and Overview • A sample of liquid protein standard 2 mg/ml Albumin from bovine serum (Sigma) was diluted 1/4 in 100 mM Tris HCl pH 8.5 . • A lyophilized Trypsin from porcine pancreas 20 µg(Sigma) was reconstituted in 20 µL of 1 mM HCl. • 1 mL of the diluted protein was added to the 20 µL of Trypsin solution and vortexed. • This sample was then transferred to and autosampler vial and put on the tray set to 36 0 C. • The sample was injected at 9 time points over the next 21 hours with tray shacking at each point. • For most time points the ultra RS diverter valve was used to prevent the large salt content from entering the CAD. • A portion of the results are discussed in the following slides. Proprietary & Confidential 2
Method Conditions • Column 1: Acclaim RSLC PAII 2.2 µm 2.1 x 250 • Column Temperature: 40 o C • Mobile Phase A: 0.1% TFA in DI Water • Mobile Phase B: 0.1% TFA in Acetonitrile • Injection Volume: 20 µL Injection Volume: 20 µL • Flow Rate: 0.75 mL/min • Gradient: Left Pump (Inverse) Right Pump Time % A % B Time % A % B 0 2 98 0 98 2 0.95 2 98 10 50 50 10.95 50 50 15 50 50 15.95 50 50 18 98 2 18.95 2 98 23 98 2 23 2 98 Proprietary & Confidential 3
Protein sample after 18 hrs with and without trypsin 35.0 1 - tryptic digest_071311 #25 [modified by lab122] Time = 18hr CAD_1 pA Az State = Off State = On 34 - 5 - FilterConstant = 3, DetectorFlowS FilterConstant = 3, DetectorFlowS 4 25.0 1 - Glucose - 1.7 29 -30 - 11 - 28 - 35 - 38 - 12 - 24 - 40 - 41 - 32 - 36 - 13 - 14 - 25 - 43 - 20 - 10 - 21 - 49 - 12.5 19 - 22 - 27 - 37 - 39 - 15 - 23 - 33 - 44 - 6 - 7 - 26 - 31 - 47 - 42 - 8 - 9 - 2 - 46 - 48 - 5 - 3 - 4 - 16 - 18 - 7 - 1 1 -5.0 75 2 - tryptic digest_071311 #32 No Trypsin CAD_1 pA 1 - Az 0 2 min -50 0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.0 21.3 23.0 Proprietary & Confidential 4
Sample with CAD and UV @ 254 and 214 nm 28.8 1 - tryptic digest_071311 #25 [modified by lab122] Time = 18hr CAD_1 pA Az DetectorFlowState = Off DetectorFlowState = On 12.5 FilterConstant = 3, FilterConstant = 3, 1 -2.6 25.0 2 - tryptic digest_071311 #25 Time = 18hr UV_VIS_1 mAU WVL:254 nm 0.0 2 -15.0 300 3 - tryptic digest_071311 #25 [modified by lab122] Time = 18hr UV_VIS_2 mAU WVL:214 nm 3 0 min -200 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 12.6 Proprietary & Confidential 5
Improved sensitivity for small peptides with CAD 16.3 1 - tryptic digest_071311 #25 [modified by lab122] Time = 18hr CAD_1 pA 10.0 1 -3.1 7.1 2 - tryptic digest_071311 #25 Time = 18hr UV_VIS_1 mAU WVL:254 nm 0.0 2 -4.6 79 3 - tryptic digest_071311 #25 [modified by lab122] Time = 18hr UV_VIS_2 mAU WVL:214 nm 3 min -21 1.23 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.18 Proprietary & Confidential 6
7 -300 250 600 2 - tryptic digest_071311 #34 110 1 - tryptic digest_071311 #34 -10 Fraction Collection using the CAD diverter valve 50 0 -0.1 mAU pA Proprietary & Confidential 2 1 w, Wvl = 214.0, B Bw = 4, RefWavelength = Off, RefBan ndwidth = 1, Az Function = 1.00 0, Data_Collection_Rate = 30, FilterC Constant = 3, Az Az FilterConstant = 3, DetectorF FlowState = Off 1.0 FilterConstant = 3, DetectorF FlowState = On 1 - Glucose - 1.7 2 - 2.0 3 - MOPS - 2.0 4 - 1 - 5 - 2 - - 6 - 3.0 3 - - 7 - 8 - 9 - 10 - 4 - - 11 - 5 - -- 12 - 6 13 - 7 - - - 14 - 8 15 - 16 -17 -18 - 9 - 4.0 1 0 - 11 - 19 - FilterConstant = 3, DetectorF FlowState = Off 20 - 21 - 22 - FilterConstant = 3, DetectorF FlowState = On 23 - 12 - 5.0 24 - 13 - 25 - 26 - 27 - 28 - 29 - 30 - 31 - 14 - 32 - 15 - 6.0 33 - 34 - Time = 21hr Time = 21hr 16 - 35 - 17 - 36 - 3 18 - 37 - 38 - 19 - 39 - 20 - 2 40 - 21 - 41 - 42 - 22 - 43 - 7.0 44 - 23 - 45 - 46 - 24 - 47 - 25 - 26 - 48 - 49 - 27 - 50 - 51 - 52 - 28 - 29 - 53 - 8.0 54 - FilterConstant = 3, DetectorF FlowState = Off 55 - 56 - 30 - FilterConstant = 3, DetectorF FlowState = On 57 - 31 - 58 - 32 - 59 - 33 - 60 - 34 - 61 - 62 - 63 - 35 5 - 9.0 64 - 65 - 36 - 66 - 37 - 67 - 68 - 38 - 69 - FilterConstant = 3, DetectorF FlowState = Off 70 - 39 9 - FilterConstant = 3, DetectorF FlowState = On 10.0 71 - 4 0 - 72 - 73 - 74 - 41 - 75 - 76 - 77 - 42 - 4 11.0 78 - 4 3 - 79 - 44 - 12.0 WVL:214 nm UV_VIS_2 CAD_1 min 12.7
Reinjection of fraction 3 18.9 1 - tryptic digest_071311 #36 Fraction #3 CAD_1 pA Az 1 - 10.0 1 0.0 -5.7 107 2 - tryptic digest_071311 #36 [modified by CCrafts] Fraction #3 UV_VIS_2 mAU WVL:214 nm 1 - 50 2 min -11 0.1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 12.8 Proprietary & Confidential 8
Conclusion • The UV @ 214 nm and the CAD show similar sensitivity to the majority of the peptides which resulted from this tryptic digest. • While the CAD is not a replacement for well established UV techniques it does provide a strong complementary technique to those already in place. • One of the largest benefits is that the sensitivity does not decrease for the smaller peptides and free amino acids which elute early in these results. • The ultra RS diverter valve also worked well to not only divert the salt S void but also collect a peak for further analysis without the need for additional plumbing or delay calculations. Proprietary & Confidential 9
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