GELFrEE1 affords rapid mass-based protein separation over a range 10-150 kDa. Here, we demonstrate a multiplexed design enabling increased loading capacity and throughput. We demonstrate comprehensive analysis of the yeast proteome using GELFrEE coupled to LC-MS/MS analysis.

1. contact: jtran@dal.ca DEPARTMENT OF CHEMISTRY, DALHOUSIE UNIVERSITY, UNIVERSITY AVENUE, HALIFAX, NOVA SCOTIA,CANADA
Department of
Chemistry
Multiplexed Gel-Eluted Liquid Fraction Entrapment Electrophoresis: A Liquid-Phase Method for Mass Separation and Analysis
John C. Tran and Alan A. Doucette
OVERVIEW
A multiplexed GELFrEE device is
reported for proteome prefractionation
8-fold increase in loading capacity &
throughput
LC-MS/MS of 16 resulting fractions
gave 420 unique yeast proteins
Separation is highly correlated to
mass of identified proteins
INTRODUCTION
GELFrEE1 affords rapid mass-based
protein separation over a range 10-150
kDa. Here, we demonstrate a
multiplexed design enabling increased
loading capacity and throughput. We
demonstrate comprehensive analysis
of the yeast proteome using GELFrEE
coupled to LC-MS/MS analysis.
1Tran & Doucette, Anal. Chem. 2008, 80, 1568-1573.
METHODS
Instrument design: 8 gel columns
are coupled in parallel to independent
collection chambers
All buffers and gels were prepared
according to Laemmli protocol
150 µL of sample is loaded per gel
column
Fractions were analyzed by 1D
gels or precipitated, digested and
subject to LC-MS/MS (1.5 hr gradient
5-40% ACN / 0.1% formic acid) on an
LTQ linear ion trap MS
ACKNOWLEDGEMENTS
Thanks to machinists Mike Boutilier and Rick Conrad
(Dalhousie) and Doucette Lab Members. This project was
funded by NSERC & the Dalhousie Chemistry Dept.
CONCLUSIONS
Multiplexed GELFrEE affords high throughput & loading
Compatible with LC MS/MS analysis
Collected fractions highly correlate to molecular weight in MS
384 well plate
S. cerevisiae
Urine proteome
B. subtilis
Fractions 1-16
High throughput mass fractionation
Higher resolution at 8-fold higher loading
Fraction 1-16 Fraction 1-16
Single column
High Resolution
(Overloaded!)
Low Resolution
8 pooled columns
800 µg load 100 µg / column
Fractions correlate highly with MW. 1126 yeast proteins (420 unique)
were identified using LC MS/MS analysis of the 16 fractions.
Fractions give high mass correlation in MS
#ProteinsIdentified
Fraction #
2 4 6 8 10 12 14 16
100
80
60
40
20
A schematic and photo of the GELFrEE device, showing
separation of stained protein markers in the gel columns (right).
Separation profile of select proteins by MS
2 4 6 8 10 12 14 16
0
10
20
30
40
50
2 4 6 8 10 12 14 16
0
2
4
6
8
10
12
14
16
Fraction #
#PeptidesIdentified
40 S Ribosomal (25 kDa)
Fraction #
2 4 6 8 10 12 14 16
0
10
20
30
40
Yeast ATP (81 kDa)
Fraction #
Heat Shock Protein (66 kDa)
A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516
10
100
20
40
70
(KDa)
Fraction #