Phosphoproteomics Analysis

Example


Phosphoproteomics (Phosphoproteomics.jpg)

Figure D2-3: New method for phosphoproteomics (Dr. Alexander Ivanov, HSPH Proteomics Facility and NIEHS Center)

Phosphoproteomics. This is a new initiative supported by the
Biological Analyses Service and developed by Dr. Alexander Ivanov.  It aims to adapt and improve phosphoproteomic
analysis of intracellular molecules using the recently upgraded instrumentation
of the HSPH Proteomics Facility. These analyses will allow detailed analysis of
signaling pathways in cellular responses to environmental agents. A new
approach for selective on-line enrichment of O-phosphorylated peptides followed
by nano-LC MS/MS analysis was developed. Various conditions for phosphopeptide
trapping and elution, geometry and content of trapping columns were tested.
Here we demonstrate the pilot results of the optimized analysis that included
several stages: trapping both O-phosphorylated and non-phosphorylated peptides
on a mixed-mode trapping column in on-line operation mode; cleaning analytes
from low molecular weight contaminants; elution of non-phosphorylated peptides
from the trapping column onto an analytical reversed phase capillary column,
followed by LC MS/MS analysis on the LTQ Orbitrap mass spectrometer.  In the second LC MS/MS run, fraction enriched
with phosphopeptides was analyzed after elution from the trapping column onto
the analytical capillary column.  Peak
area values corresponding to each peptide were measured using LC-MS extracted
ion chromatograms and normalized to the total peak area of the peptide detected
in both LC MS runs. The main advantage will
be the ability to completely automate, eliminate sample losses and increase
accurate throughput for enrichment and analysis of phosphorylated peptides in
samples from Center.

Graphed
in Figure D2-3:
Mass
spectrometry- based phosphopeptide analysis using the developed technique for
on-line phosphopeptide enrichment. A: extracted ion LC-MS chromatograms for the
non-retaining flow-through peptide fraction (in red) and phosphopeptide
enriched fraction (blue) demonstrate efficiency of phosphopeptide enrichment
(>99%). B: MS spectrum corresponding to the elution time of the selected
phosphopeptide. High mass accuracy high mass resolution detection of the triply
charged ion is demonstrated on the zoomed up area of the spectrum. C:[A1]  Tandem
mass spectrum corresponding to the parent ion detected in the MS spectrum shown
on panel B. Identified fragment ions shown in color and highlighted in the
table listing theoretically predicted fragments.


 [A1]does
not correspond to any labels on the figure