CHAPTER 4
Direct Analysis of Protein Complexes
J. R. YATES!, A. J. LINKl, D. SCHIELTZ!, J. K. ENG l
1
Introduction
Deciphering the functions of genes discovered by genome sequencing will be a
major challenge of the post-genome era. Physiological processes are performed
primarily by proteins and these functions are often accomplished with other proteins as components of multi-protein complexes, as part of signal transduction
pathways or as ligands for receptors. Identifying the sets of proteins involved in
a process will be key to understanding the individual roles of proteins.
Approaches to accomplish this goal will encompass at least two types of measurements. First, by measuring expression levels of proteins under different cellular
conditions and states, co-regulation of protein expression can be observed. Coregulation will not necessarily indicate proteins are part of a complex or pathway,
but that their functions are regulated as part of the process. A second measurement will encompass dissection of the components of protein complexes. Proteins perform many of their functions in concert with other proteins, by forming
stable complexes or through more subtle interactions. A protein's presence in a
complex is more reflective of direct involvement in a process then association by
co-regulation. Some proteins in complexes may be co-regulated and others may
not be. By identifying the interacting proteins as a function of cellular state,
insight into the networks of proteins involved in those processes will be obtained.
By linking this information to that obtained by expression level measurements, a
broader picture will be obtained of those proteins directly involved and those
involved in more peripheral aspects of the process. A necessary element to these
studies is the ability to rapidly identify the often-complex sets of proteins.
The capability of mass spectrometers to analyze and identify proteins and
pep tides has improved over the last few years. Refinements to ionization methods
have resulted in improved sensitivities and integration with liquid separation
methods. In combination with tandem mass spectrometers, these improvements
have created a powerful technique to sequence peptides. The fundamental process was described by Hunt et al. in the early 1980's (Hunt et al 1986). Peptide
ions are selected in the first mass analyzer and passed into a gas-phase collision
cell. Ions are activated to fragment through low-energy gas-phase collisions. The
I Department of Molecular Biotechnology, Box 357730, University of Washington, Seattle, WA
98195-7730.
Direct Analysis of Protein Complexes
J. R. YATES!, A. J. LINKl, D. SCHIELTZ!, J. K. ENG l
1
Introduction
Deciphering the functions of genes discovered by genome sequencing will be a
major challenge of the post-genome era. Physiological processes are performed
primarily by proteins and these functions are often accomplished with other proteins as components of multi-protein complexes, as part of signal transduction
pathways or as ligands for receptors. Identifying the sets of proteins involved in
a process will be key to understanding the individual roles of proteins.
Approaches to accomplish this goal will encompass at least two types of measurements. First, by measuring expression levels of proteins under different cellular
conditions and states, co-regulation of protein expression can be observed. Coregulation will not necessarily indicate proteins are part of a complex or pathway,
but that their functions are regulated as part of the process. A second measurement will encompass dissection of the components of protein complexes. Proteins perform many of their functions in concert with other proteins, by forming
stable complexes or through more subtle interactions. A protein's presence in a
complex is more reflective of direct involvement in a process then association by
co-regulation. Some proteins in complexes may be co-regulated and others may
not be. By identifying the interacting proteins as a function of cellular state,
insight into the networks of proteins involved in those processes will be obtained.
By linking this information to that obtained by expression level measurements, a
broader picture will be obtained of those proteins directly involved and those
involved in more peripheral aspects of the process. A necessary element to these
studies is the ability to rapidly identify the often-complex sets of proteins.
The capability of mass spectrometers to analyze and identify proteins and
pep tides has improved over the last few years. Refinements to ionization methods
have resulted in improved sensitivities and integration with liquid separation
methods. In combination with tandem mass spectrometers, these improvements
have created a powerful technique to sequence peptides. The fundamental process was described by Hunt et al. in the early 1980's (Hunt et al 1986). Peptide
ions are selected in the first mass analyzer and passed into a gas-phase collision
cell. Ions are activated to fragment through low-energy gas-phase collisions. The
I Department of Molecular Biotechnology, Box 357730, University of Washington, Seattle, WA
98195-7730.