| Two-dimensional HPLC/MS has been widely
used for proteomic analyses of proteolytic hydrolysates
of complex protein mixtures because it combines high capacity
chromatographic separation with exquisite electrospray
ionization efficiency and the selectivity of tandem mass
analyses. The ability to identify and characterize low
abundance proteins in the presence of a background of
common, high copy number proteins remains a significant
problem in analytical biochemistry. Many 2D chromatographic
separations for peptides have been described. However,
issues of loading capacity, dynamic range, and sample
pre-treatment requirements may preclude their practical
application.
We evaluated the dynamic range of a 2D HPLC system
to be more tolerant of peptide digest solutions for
routine use without sample pretreatment. This 2D HPLC
system was developed in a Cooperative Research and Development
Agreement between NIH and Shimadzu and combines a high
capacity ion exchange column with a capillary reversed
phase column. A capillary trapping column compatible
with variable flow rates links both separation columns.
The automated system provides high loading capacity
and high sensitivity, and serves as a robust platform
for high throughput proteomics analyses.
Primary evaluation of the dynamic range was performed
using tryptic digests of standard proteins. Mixtures
containing equimolar and low concentration (1% relative
to major component) proteins were analyzed. Discrete
SCX gradient steps (either 3, 6, or 15) were tested
to determine the fractionation of identified analytes
eluted from the SCX column. Using a three-step SCX gradient,
three of the five low abundance species, were identified
in a Mascot database search report, while all five of
the 1% proteins were identified in six or fifteen step
gradient elutions.
These results indicate the combination of high capacity
SCX column and multiple sequential step fractionation
followed by capillary RP nanospray ESI-MS enables the
detection of low abundance proteins in complex mixtures
with increased dynamic range and selectivity. The effect
of the number of fractions and sample pre-treatment
will be also described. |
