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Quantitative Proteomics Center
Our Synapt G2 QTOF mass spectrometer with ion mobility separation and NanoAcquity UPLC
(Waters Corp.) allow us to implement label-free protein profiling, a
leading technique in quantitative discovery proteomics for systems
biology and biomarkers. Drosophila, yeast, E. coli, Neisseria, Nitrosomonas, mouse, rat and human and viral proteomes have been studied in our lab.
1. W. W. Wu, G. H. Wang, S. J. Baek, R. F. Shen, Journal Of Proteome Research 5, 651 (Mar, 2006).
2. J. C. Silva et al., Molecular & Cellular Proteomics 5, 589 (Apr, 2006).
3. J. P. C. Vissers, J.
I. Langridge, J. Aerts, Molecular & Cellular Proteomics 6, 755 (May, 2007).
4. D. M. Xu et al., Molecular & Cellular Proteomics 7, 2215 (Nov, 2008).
5. J. C. Silva, M. V.
Gorenstein, G. Z. Li, J. P. C. Vissers, S. J. Geromanos, Molecular
& Cellular Proteomics 5, 144 (Jan, 2006).
6. F. Y. Cheng, K.
Blackburn, Y. M. Lin, M. B. Goshe, J. D. Williamson, Journal Of
Proteome Research 8, 82 (Jan, 2009).
7. X. F. Wang et al., Developmental Cell 15, 220 (Aug, 2008).
Label-Free Shotgun Proteomic Profiling
quantitative protein abundance data from adult human stem cells shows
distinct pattern of expression in cluster analysis
We use splitless nanoflow
chromatography coupled with quadrupole time-of-flight (QTOF) mass
spectrometry to determine proteins quantities in cells, tissues,
subcellular fractions and affinity fractions, biofluids (serum, plasma,
urine and saliva proteomics). Ion mobility separation adds a
multidimensional (2D) separation based on shape and cross sectional
area of peptides in addition to mass/charge ratio.
In one study of protein levels in a model system for apoptosis and
regeneration in the mouse olfactory epithelium we found differences in
protein levels for numerous proteins in the comparison of treatments
affecting these biological processes. The validity of these results and
the overall utility of this technique were demonstrated by the fact
that five already known markers of these processes were confirmed by
this technique and a number of new proteins were discovered as
In another study with E. coli we confirmed differential
regulation of several proteins already detected by the DIGE technique
and other methods, and also found new differentially-regulated proteins
in this gene knockout experiment.
Recent innovations in this area have demonstrated a variant of the
label-free approach in which mass spectra are recorded at alternate low
(precursor) and high (product) fragmentation voltages in a method
called MSE (2).
In this approach rapidly alternating parent and product ion spectra are
generated in a protocol that seeks to record "all the ions all the
time, and these data are analyzed with an ion accounting
algorithm. Increases in reproducibility of chromatographic separations
have enhanced the feasibility of this approach. Building on these
conceptual and instrumentation advances a number of groups have
demonstrated effective use of this method for microbial cells (2), human serum (3), and cancer cells (4). Waters Corporation (Milford, MA) has implemented the MSE algorithm in a software routine known as IdentityE. We have used this system to study proteomes of Drosophila, yeast, bacterial (E. coli, Neisseria, Nitrosomonas), mouse, rat and human proteomes.
We use two complementary techniques to
compare different proteomes or compare affinity purifications and
immunoprecipitates. The first is label-free shotgun protein
profiling as described here. Another technique called difference
gel electrophoresis (DIGE) is also described in this website (click here).
Background in Quantitative Proteomic Analysis
Early approaches to quantitative proteomics used a comparison of
2-dimensional electrophoretic gels (2D gels) coupled with mass
spectrometry, then progressed to the use of isotopic labeling (e. g.
ICAT, iTRAQ, SILAC) in a shotgun liquid chromatography/mass
spectrometry (LC-MS and LC-MSMS) strategy, or the use of fluorescence
labeling in gel-based proteomics (1).
All of these approaches have some limitations. The gel-based approaches
tend to be cumbersome to use, place restrictions on experimental design
and address a limited subset of proteins. The isotopic methods also
place stringent limits on experimental design, the type of samples that
may be analyzed, and often rely on weak isotopic signatures. Protein
arrays are at an early stage of development. Finally, targeted
(multiple reaction monitoring mass spectrometry) analyses address a
limited pre-determined selection of proteins and can be expensive to
implement. Consequently, there is increasing interest in using
so-called "label-free methods to make proteome-wide comparisons.
This technique is an ideal discovery tool for understanding biological
processes, stem cells, developing biomarkers for a variety of uses, comparing affinity purifications and immunoprecipitations and studying biofluids such as urine, saliva, plasma and serum.