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

OVERVIEW 3D SEPARATION PLATFORM 2D LE COUPLED TO LC-MS/MS

6903.0 Da
y17 y11
b31
¾ 3D Top Down proteomics separation platform Solution Multiplex Capillary b5 b30
10819.2 Da
incorporates solution isoelectric focusing (sIEF), multiplex gel-
eluted liquid fraction entrapment electrophoresis (mGELFrEE), IEF 8 GELFrEE 8 × 14 RPLC 14094.0 Da
15432.1 Da

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

throughput protein identifications. 15251.9 Da

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-

simultaneously fractionated by multiplex gel-eluted liquid fraction entrapment electrophoresis -R-R-S-Y-F-H-E-D-D-K-F-I-A-D-K-T-K-F-A-L-G-Q-G-V-G-V-I-L-C-I-G-E-T-

-L-E-E-K-K-A-G-K-T-L-D-V-V-E-R-Q-L-N-A-V-L-E-E-V-K-D-W-T-N-V-V-V-A-
The emerging field of high-throughput Top Down proteomics into 14 liquid fractions according to size. These 112 fractions were precipitated, resuspended -Y-E-P-V-W-A-I-G-T-G-L-A-A-T-P-E-D-A-Q-D-I-H-A-S-I-R-K-F-L-A-S-K-L-
-G-D-K-A-A-S-E-L-R-I-L-Y-G-G-S-A-N-G-S-N-A-V-T-F-K-D-K-A-D-V-D-G-F-
has brought a renewed demand for high quality intact protein and subjected to LC MS/MS analysis. -L-V-G-G-A-S-L-K-P-E-F-V-D-I-I-N-S-R-N

separations in the solution phase. To ensure maximum peak

capacity, multidimensional separations are commonly 22 24 26 28 30 32 34
Time (min) MS/MS spectra of Triosephosphate Isomerase
employed for complex proteomes. However, although the
combination of more than two separation modes is desirable, 2D LIQUID ELECTROPHORESIS (2D LE) RPLC chromatogram of IEF Fr.1 - GELFrEE Fr.1. (26.6 kDa). Prosight output shows 58 matching
Isotopic distributions for selected proteins detected are fragments with E-Value 9×10-50.
it remains rare in proteomics due to many difficulties. This Isoelectric Focusing
ladder

shown.
kDa

work covers details of our three-dimensional separation 2

1 2
pH 12
3 4 5 6 7 8 std
Gel-Eluted Liquid Fraction Entrapment Electrophoresis
100
platform with online detection and identification using the high 75
sIEF Fr.1 sIEF Fr.2
1 GELFrEE Fr. 14 1 GELFrEE Fr. 14
resolution 12T-LTQ-FT mass spectrometer.1 This work 50

represents the first reported semi-preparative multiplexed

37
25
3D SEPARATION DISPLAY IMAGE
20 10 70 min
two-dimensional liquid electrophoretic platform2,3 that has 15 40 kDa
ever been coupled to LC-LTQ-FTMS; thereby affording 10
comprehensive separations and unambiguous identifications 10 kDa
and characterization of proteins from complex biological 9 35

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

focused for 1.5 h at 2W. The sample was acetone 10

precipitated to 50 µL and simultaneously separated with
mGELFrEE.
¾ mGELFrEE: The eight 15%T polyacrylamide parallel
gel columns are interfaced in parallel to independent
collection chambers. All buffers and gels were prepared
according to Laemmli protocol. 50 μL of sample was loaded
per gel column. Separation occurs simultaneously requiring
1.5 h at 240V.
¾ LC-MS/MS analysis: 2D LE fractions were precipitated,
and subject to a 75 µm i.d. × 10 cm PLRPS (5 um, 1000 Å)
column. LC-MS/MS (40 min gradient 30-55% ACN/0.2% FA)
was operated on an 12T-LTQ-FT ultra mass spectrometer.
1 2 3 4 5 6 7 8
IEF FRACTION
Gel image of 8 fractions collected from 1.5 hr sIEF separation of 4 mg yeast protein
(highlighted in red). These eight fractions were further separated using mGELFrEE. Gel Display image shows heat map of protein detection from the 3D separation (IEF Fr. 1-8 and GELFrEE
images showing the 2nd dimension separation of the fractions collected using mGELFrEE. Fr. 3-10) (left). Each grid corresponds to a 2D LE fraction, with RPLC elution time (10-70 min) versus
All eight sIEF fractions from the 1st dimension (left panel) were simultaneously separated in average mass (10-40 kDa). Average mass was deconvoluted through selected ion chromatographic
1.5 hours. Total 2D LE separation took 3 hcovering 2<pH<12 and 10<MW<75 kDa. profiles of all detected masses. Insets show zoom in plots of a low and high MW GELFrEE Fraction.
ACKNOWLEDGEMENTS CONCLUSIONS
Thanks to Ioanna Ntai, Mingxi Li, Steve Sweet, Dorothy Alhf, Haley Thomas, Brad Evans, Stephanie Bumpus, Yunqiu Chen, ¾ High resolution separation simplifies proteome complexity.
Jeremiah Tipton, Chris Hendrickson, Alan Marshall, Alan Doucette, Robert Guy, Louis Ramaley and former Kelleher and Doucette ¾ 2D LE is compatible with LC MS/MS Top Down analysis.
Lab Members. This project was funded by National Institutes of Health GM 067193-07 and the Roy J. Carver Charitable Trust. ¾ Proteome profile heat maps enable differential mapping.