Genomic Health Evaluation of Genomic Health Evaluation of Corona - - PowerPoint PPT Presentation

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 0C.

• 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.

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Method Conditions

• Column 1: Acclaim RSLC PAII 2.2 µm 2.1 x 250
• Column Temperature: 40oC
• 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:

Time % A % B 98 2 Right Pump Time % A % B 2 98 0.95 2 98 Left Pump (Inverse) 10 50 50 15 50 50 18 98 2 23 98 2 10.95 50 50 15.95 50 50 18.95 2 98 23 2 98

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Protein sample after 18 hrs with and without trypsin

35.0 1 - tryptic digest_071311 #25 [modified by lab122] Time = 18hr CAD_1 pA

34 - 5 -

Az State = Off State = On

12.5 25.0

1 - Glucose - 1.7 2 - 3 - 4 - 5 - 6 - 7 - 8 - 9 - 10 - 11 - 12 - 13 - 14 - 15 - 16 - 7 - 18 - 19 - 20 - 21 - 22 - 23 - 24 - 25 - 26 - 27 - 28 - 29 -30 - 31 - 32 - 33 - 35 - 36 - 37 - 38 - 39 - 40 - 41 - 42 - 43 - 44 - 4 46 - 47 - 48 - 49 -

FilterConstant = 3, DetectorFlowS FilterConstant = 3, DetectorFlowS

• 5.0

1 1

75 2 - tryptic digest_071311 #32 No Trypsin CAD_1 pA

1 -

Az

0 2 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

• 50

min

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Sample with CAD and UV @ 254 and 214 nm

12.5 28.8 1 - tryptic digest_071311 #25 [modified by lab122] Time = 18hr CAD_1 pA

Az DetectorFlowState = Off DetectorFlowState = On

• 2.6

1

FilterConstant = 3, FilterConstant = 3,

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

• 200

min

3

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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

Improved sensitivity for small peptides with CAD

10.0 16.3 1 - tryptic digest_071311 #25 [modified by lab122] Time = 18hr CAD_1 pA

1

• 3.1

7.1 2 - tryptic digest_071311 #25 Time = 18hr UV_VIS_1 mAU WVL:254 nm

• 4.6

0.0 2 79 3 - tryptic digest_071311 #25 [modified by lab122] Time = 18hr UV_VIS_2 mAU WVL:214 nm 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

• 21

min

3

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Fraction Collection using the CAD diverter valve

110 1 - tryptic digest_071311 #34 Time = 21hr CAD_1 pA

Constant = 3, Az Az FlowState = Off FlowState = On FlowState = Off FlowState = On FlowState = Off FlowState = On FlowState = Off FlowState = On

50

• -
• 0 -

11 - 12 - 13 - 14 - 15 - 16 - 17 - 18 - 19 - 20 - 21 - 22 - 23 - 24 - 25 - 26 - 27 - 28 - 29 - 30 - 31 - 32 - 33 - 34 - 5 - 36 - 37 - 38 - 9 - 0 - 41 - 42 - 3 - 44 -

0, Data_Collection_Rate = 30, FilterC FilterConstant = 3, DetectorF FilterConstant = 3, DetectorF FilterConstant = 3, DetectorF FilterConstant = 3, DetectorF FilterConstant = 3, DetectorF FilterConstant = 3, DetectorF FilterConstant = 3, DetectorF FilterConstant = 3, DetectorF

• 10

1 1 - 2 - 3 - 4 - 5 - 6 7 - 8 9 1 2 35 39 4 4 4

Function = 1.00

600 2 - tryptic digest_071311 #34 Time = 21hr UV_VIS_2 mAU

36 - 52 - 69 -

WVL:214 nm

ndwidth = 1, Az

250

2 1 - Glucose - 1.7 2 - 3 - MOPS - 2.0 4 - 5 - 6 - 7 - 8 - 9 - 10 - 11 - 12 - 13 - 14 - 15 - 16 -17 -18 - 19 - 20 - 21 - 22 - 23 - 24 - 25 - 26 - 27 - 28 - 29 - 30 - 31 - 32 - 33 - 34 - 35 - 3 37 - 38 - 39 - 40 - 41 - 42 - 43 - 44 - 45 - 46 - 47 - 48 - 49 - 50 - 51 - 53 - 54 - 55 - 56 - 57 - 58 - 59 - 60 - 61 - 62 - 63 - 64 - 65 - 66 - 67 - 68 - 70 - 71 - 72 - 73 - 74 - 75 - 76 - 77 - 78 - 79 -

Bw = 4, RefWavelength = Off, RefBan

• 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 12.7

• 300

min

w, Wvl = 214.0, B

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Reinjection of fraction 3

18.9 1 - tryptic digest_071311 #36 Fraction #3 CAD_1 pA

1 -

Az

0.0 10.0

1

• 5.7

107 2 - tryptic digest_071311 #36 [modified by CCrafts] Fraction #3 UV_VIS_2 mAU

1 -

WVL:214 nm 50 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

• 11

min

2

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Conclusion

• The UV @ 214 nm and the CAD show similar sensitivity to the majority
• f 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. S

• The ultra RS diverter valve also worked well to not only divert the salt

void but also collect a peak for further analysis without the need for additional plumbing or delay calculations.

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