powerpoint presentationgenomics.unl.edu/rbc_2014/course_files/tue5.pdfanalyze by hpcl em: 420 ....
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Sample 1
ICAT light
ICAT heavy
TCEP Red
Avidin Selec
Trypsin Dig
GIST light
Sample 2
ICAT light
ICAT heavy
TCEP Red
Avidin Selec
Trypsin Dig
GIST heavy Mix
LC-MS(/MS)
Output: four co-eluting peeks representing redox status and two samples Peak 1: Ox C in S1 Peak 2 (+3Da): Ox C in S2 Peak 3 (+9Da): Red C in S1 Peak 4 (+12Da) Red C in S2
Double Labeling in Global Thiol Redox Proteome
1 4
2
3
5
Carbonyl Content Using Cy3 and Cy5 - hydrazide Chemistry for
2D ELFO
Sample 1
Cy3 hyrdazide
Sample 2
Cy5 hydrazide
Mix
2D ELFO and analysis
Sample, extracted in phosphate buffer, then MeOH precipitated
+
+
+
TCEP (Heavy GSH)
Add diluted 2-VPd7, heavy label reduced GSH
Add excess light 2-VP, then TCEP, light label GSSG (reduced by TCEP) Can add isotopic labeled GSH at this step for absolute Quantification
Analysis of GSH/GSSG content
Method 6 - this method was currently developed at Adamec’s lab and utilizes light/heavy 4-VP for GSH derivatization. Tagged
Red/Ox GSH is quantified using HPLC (C18) coupled with FTICR.
GSH/GSSG content - Result
0 2 4 6 8 10 Time [min]0
1
2
3
4
5
7x10
Intens.
2_7-24_1-b,2_01_576.d: EIC 413.1±0.1 +All MS, Smoothed (0.88,1,GA) 2_7-24_1-b,2_01_576.d: EIC 416.1±0.1 +All MS, Smoothed (0.88,1,GA)
2_7-24_1-b,2_01_576.d: EIC 420.1±0.1 +All MS, Smoothed (0.88,1,GA)
0 2 4 6 8 10 Time [min]0
2
4
6
9x10
Intens.
14a.d: TIC +All MS
m/z 413.15 GSSG 416.15 Internal Standard GSH 420.15 GSH
Adenine Containing Molecules: Derivitize with
Chloroacetaldehyde
Production of etheno adducts
EX: 300 EM: 420 Analyze by HPCL
Standard Compounds
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 min0
25000
50000
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150000
175000
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225000
250000
275000
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325000
350000
375000
400000
425000
450000uV