Title

The availability of the genome database, the evolution of high-resolution protein separation techniques and mass spectrometers for rapid identification of proteins have converged to re-define the kinds of questions one can ask of biological systems, as well as the time-frames involved. From the studies published thus far utilizing genomic and proteomic approaches, it is clear that biological events initiated with single gene or protein events ultimately involve changes in multiple genes and proteins. This tutorial will review the principal proteomics methods that resolve proteins in complex mixtures for mass spectrometric analysis. Specifically, the two-dimensional electrophoretic methods, isoelectric focussing/SDS-PAGE (IEF/SDS-PAGE), and 2-D "blue-native" (2D-BN) electrophoresis will be discussed.  In IEF/SDS-PAGE, proteins are separated initially according to charge differences, and secondly according to size differences. In 2D-BN, polypeptides are separated in a non-denaturing first dimension as complexes. These are resolved into their components in the second dimension by standard SDS-PAGE. The advantages and limitations of both methodologies will be discussed, as well as experimental parameters that affect detection and resolution of proteins in both types of 2-DE. Results obtained by the author using these proteomics approaches will be shared.

"Homogenous Affinity Arrays And Assays: Quantitative Proteomics On A Chip" by Elaine Scrivvener, Oxford Glycosciences 
Elaine.scrivener@ogs.co.uk

Characterization of the complement of expressed 
proteins from a single genome is a central focus of the 
evolving field of proteomics. Monitoring the expressions 
and properties of a large number of proteins provides 
important information about the physiological or 
biochemical state of a cell. Since a cell can express a 
large number of different proteins, the characterization of 
thousands of proteins can only be readily accomplished 
using a high-throughput, process. Microarray formats for 
the quantitative detection of proteins have been developed 
for diagnosis and protein-protein interaction discovery. 
However, development of a high complexity microarray 
affinity capture system for semi-quantitative or quantitative 
proteomics similar to those used for semi-quantitative 
mRNA expression analyses is more complex. Nucleic 
acids serve as information carriers, but also embody 
heterogeneous affinity capture agents and exhibit similar 
affinities for each target. Unlike nucleic acids, proteins 
are extremely heterogeneous, e.g., small and soluble 
proteins, large and highly post-translationally modified 
proteins, neutral or highly charged proteins and proteins 
with multiple domains of varying hydrophilicity (e.g. 
transmembrane receptors) etc. This means that an array 
affinity capture system for semi-quantitative or quantitative 
proteomics requires extremely extensive optimisation of 
capture affinity reagent-protein interactions for a format to 
be achieved in which each affinity capture agent interacts
with its corresponding target protein or family of target 
proteins in a uniform and predictable fashion. We have 
developed a potential solution to the protein heterogeneity 
problem. Our methods result in a more homogenous 
assay format and allow simultaneous quantitative analysis 
of expression of proteins of heterogeneous nature.

"Adding Meaning to Complex Sets; Implementation of Multi-Tiered Database Searching And Spectrum Interpretation In A Multidimensional LC/MS/MS Experiment" by Roy Martin, Micromas
Roy_Martin@mspeople.com

Analysis of digested mixtures of proteins by two-dimensional LC/MS/MS generates very large numbers of peptide MS/MS spectra that are intended to identify the proteins.  One of the problems of this approach is that the high dynamic range of these experiments means that there are preferentially many intense spectra of higher abundance and few, weaker spectra from the lower and often more interested proteins the mix. Also, the presence of so many spectra often means that there is little time available to monitor each and every one of the database hits for quality, and to further try to interpret unmatched spectra for new sequences and possible modifications not predicted in the database search. In this presentation we describe the methods for generating large data sets and then analyzing the spectra that are generated. Data are analyzed by a tiered series of algorithms; each adding more information that first increases the confidence of a database hit and then adds new information. After the initial pass against a database, spectra are passed through an algorithm that searches for possible modifications.  Remaining high quality spectra can be de novo sequenced and BLAST searched.  The results are displayed in a comprehensive form that brings together all the information derived on a protein in the mixture.

"New Technology For An Old Disease: Using 
ProteinChip® Arrays To Investigate Pre-eclampsia" 
by Stella Redpath, Ciphergen
sredpath@ciphergen.com

Preeclampsia is a pregnancy condition with a multi-factorial etiology, first described more than one century ago, but still the molecular and cellular events underlying the disease are unknown.  We hypothesize that the key molecular features of this complex disorder lie in abrogated events at the maternal-fetal interface and thus inadequate placentation. We investigated preeclampsia using Ciphergen Biosystem ProteinChip® arrays and surface-enhanced laser desorption/ionization (SELDI®) to characterize protein expression profiles of placental tissue.  Proteomics data could lead to a new  understanding of preeclampsia. Using SELDI® technology, we demonstrate that analysis of protein expression profiles of placental and fetal membranes may be a rapid approach to search for candidate biomarkers expressed during preeclampsia.  In this pilot study, we present data representing a first global assessment of the placental and fetal membranes at the protein level.