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2D Fractionation of Intact Proteins Prior To RPLC Coupled To LTQ-FT MS/MS
2D Fractionation of Intact Proteins Prior To RPLC Coupled To LTQ-FT MS/MS
2D Fractionation of Intact Proteins Prior To RPLC Coupled To LTQ-FT MS/MS
John C. Tran, Cong Wu, John F. Kellie, Ji Eun Lee, Adaikkalam Vellaichamy, Kenneth R. Durbin, Adam D. Catherman, Leonid Zamdborg, Paul M. Thomas, Neil L. Kelleher
contact: johntran@illinois.edu DEPARTMENT OF CHEMISTRY, UNIVERSITY OF ILLINOIS, SOUTH MATHEWS AVENUE, URBANA, ILLINOIS, USA
and capillary reversed phase liquid chromatography (cRPLC) Fr.actions Fractions b31
b82
11165.0 Da
y71
¾ Two dimensional liquid electrophoresis (2D LE) affords 8 13529.1 Da
11123.3
sIEF fractions that are simultaneously separated with Da y5 y14
b37
b34y18 y71
b36
mGELFrEE (14 fractions) resulting in 112 resolved fractions b35b29 y
7 y74
8850.5 Da
covering 2<pH<12 and 5-75 kDa in 3 h. y11
b27 b4
b16 b35 b37b
38
y73
y72
y16 y72 y71
¾ 2D LE fractions coupled to LC MS/MS enables high 14808.6 Da
Y8 b9
y19 b33
y76
12375.6 Da
500 600 700 800 900 1000 1100 1200 1300 1400
m/z
INTRODUCTION Solution isoelectric focusing affords 8 fractions resolved by pI. These eight fractions are
-A-R-T-F-F-V-G-G-N-F-K-L-N-G-S-K-Q-S-I-K-E-I-V-E-R-L-N-T-A-S-I-P-E-
-N-V-E-V-V-I-C-P-P-A-T-Y-L-D-Y-S-V-S-L-V-K-K-P-Q-V-T-V-G-A-Q-N-A-Y-
-L-K-A-S-G-A-S-G-A-F-T-G-E-N-S-V-D-Q-I-K-D-V-G-A-K-W-V-I-L-G-H-S-E-
shown.
kDa
kDa
sIEF Fr.3 sIEF Fr.4 sIEF Fr.5
samples in a highly automated and high throughput fashion.
GELFrEE FRACTION
100 1 GELFrEE Fr. 14 1 GELFrEE Fr. 14 1 GELFrEE Fr. 14 30
75
50
8
1. M.J. Roth, B.A. Parks, J.T. Ferguson, MT. Boyne, N. L. Kelleher, Anal. Chem. 2008, 80, 2857-2866. 37
2. J.C. Tran, A.A. Doucette, J. Proteome Res., 2008, 7, 1761-1766. 25
3. J.C. Tran, A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573. 20 7 35 40 45 50 55 60 65
15
10 6
sIEF Fr.6 sIEF Fr.7 sIEF Fr.8 15
GELFrEE Fr. GELFrEE Fr. GELFrEE Fr. 5
METHODS 100 1 14 1 14 1 14
kDa
75
50
37 4 10
25
¾ sIEF: 4 mg yeast (8M urea, 50 mM DTT, 1% 3/10 20
3
30 35 40 45 50 55
RPLC Time (min)
biolyte) was loaded into 8 chambers (400 uL each) and 15