Strategies and Methods for Proteome Analysis
5
as for single, isolated proteins. We will discuss current limitations in the technology, tips for avoiding pitfalls and circumventing problems. Specifically we hope
to provide the reader with an overview and helpful hints on the following subject
matter:
- Global Protein Separation
- Sample Preparation for Mass Spectrometry
- Sample Introduction for Mass Spectrometry
- Protein Identification by Tandem Mass Spectrometry
- Challenges of Low Abundance Proteins
- Phosphopeptide Analysis.
1
Global Protein Separation
The method of choice for separation of complex protein mixtures is polyacrylamide gel electrophoresis (PAGE). Separation by PAGE not only provides resolving power but also facilitates subsequent proteolytic digestion because PAGE
most frequently is done in a denaturing environment. Partially purified samples
of limited complexity can be efficiently separated by one-dimensional PAGE. For
complex mixtures such as whole celllysates a two-dimensional (IEF/SDS-PAGE
or 2DE) separation is usually more suitable. The 2DE method has the potential to
separate several thousand proteins (Gorg, et al. 1988; Klose and Kobalz, 1995) in
a single experiment and therefore provides the opportunity to detect differences
in protein expression between two or more samples by comparative analysis of
global protein patterns. 2DE is currently the protein separation method on which
proteome analysis is based.
Visualization of proteins in gels is usually by Coomassie blue or silver staining
or if the protein is radiolabeled by autoradiography. It is difficult to predict a priori which stained protein will be identifiable by MS/MS, but some generic guidelines can be stated. In general a protein visible by Coomassie blue can be easily
identified by the methods outlined in this manuscript. Whereas a protein visible
as a very faint silver stained spot «5 ng of protein) mayor may notbe identifiable unless multiple, identical spots from separate gels are pooled. Regulatory
proteins or other proteins present in cells at low abundance are not visible by silver staining of total cell lysates and are therefore impossible to identify without
selective enrichment prior to gel electrophoresis.
2
Sample Preparation for Mass Spectrometry
Due to the sensitivities of current mass spectrometric techniques sample contamination is a concern. Careless sample handling or use of buffers and reagents
not optimized for high sensitivity applications (Zhang et al 1998) will inevitably
lead to contamination with proteins such as kerratin. It is therefore important to
avoid contamination by maintaining a clean work environment, wearing gloves
and using quality-assured reagents. To avoid other sources of contamination a
dedicated gel dryer and gel equipment are recommended for high sensitivity pro-
5
as for single, isolated proteins. We will discuss current limitations in the technology, tips for avoiding pitfalls and circumventing problems. Specifically we hope
to provide the reader with an overview and helpful hints on the following subject
matter:
- Global Protein Separation
- Sample Preparation for Mass Spectrometry
- Sample Introduction for Mass Spectrometry
- Protein Identification by Tandem Mass Spectrometry
- Challenges of Low Abundance Proteins
- Phosphopeptide Analysis.
1
Global Protein Separation
The method of choice for separation of complex protein mixtures is polyacrylamide gel electrophoresis (PAGE). Separation by PAGE not only provides resolving power but also facilitates subsequent proteolytic digestion because PAGE
most frequently is done in a denaturing environment. Partially purified samples
of limited complexity can be efficiently separated by one-dimensional PAGE. For
complex mixtures such as whole celllysates a two-dimensional (IEF/SDS-PAGE
or 2DE) separation is usually more suitable. The 2DE method has the potential to
separate several thousand proteins (Gorg, et al. 1988; Klose and Kobalz, 1995) in
a single experiment and therefore provides the opportunity to detect differences
in protein expression between two or more samples by comparative analysis of
global protein patterns. 2DE is currently the protein separation method on which
proteome analysis is based.
Visualization of proteins in gels is usually by Coomassie blue or silver staining
or if the protein is radiolabeled by autoradiography. It is difficult to predict a priori which stained protein will be identifiable by MS/MS, but some generic guidelines can be stated. In general a protein visible by Coomassie blue can be easily
identified by the methods outlined in this manuscript. Whereas a protein visible
as a very faint silver stained spot «5 ng of protein) mayor may notbe identifiable unless multiple, identical spots from separate gels are pooled. Regulatory
proteins or other proteins present in cells at low abundance are not visible by silver staining of total cell lysates and are therefore impossible to identify without
selective enrichment prior to gel electrophoresis.
2
Sample Preparation for Mass Spectrometry
Due to the sensitivities of current mass spectrometric techniques sample contamination is a concern. Careless sample handling or use of buffers and reagents
not optimized for high sensitivity applications (Zhang et al 1998) will inevitably
lead to contamination with proteins such as kerratin. It is therefore important to
avoid contamination by maintaining a clean work environment, wearing gloves
and using quality-assured reagents. To avoid other sources of contamination a
dedicated gel dryer and gel equipment are recommended for high sensitivity pro-