PowerPoint Presentationgenomics.unl.edu/RBC_2014/COURSE_FILES/tue5.pdfAnalyze by HPCL EM: 420 . 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

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

75000

100000

125000

150000

175000

200000

225000

250000

275000

300000

325000

350000

375000

400000

425000

450000uV

Mouse Brain Extract

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

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000

uV