Strategies and Methods for Proteome Analysis
7
Conclusions
13
While it is apparent that current 2DE technology has limitations for proteome
analysis it is nevertheless extremely useful for large scale protein analysis and for
the analysis of specific proteins in a sample. As noted by an examination of codon
bias values (Gygi et al. 1998) coverage of an entire proteome is currently out of
reach because of the low copy number of most proteins in any given cell. However, those using the technology to ask direct biological questions can be successful (Santos et al. 1998). To provide the best chance of success research questions
involving large scale protein analysis should be addressed using a suitable combination of the available biochemical and immunological tools for isolation and
purification of proteins. As for the future of mass spectrometers in proteomics it
should be possible to identify a protein based on accurate mass measurement of
a single peptide using FT-ICR mass spectrometers. Additionally, the wider use of
MALDI on instruments such as FT-ICR and ion trap mass spectrometers may
facilitate a more judicious use of valuable samples fractionated on-line during
ESI-MS/MS at nanoliter/minute flow rates and then re-analyzed on MALD!
instruments.
8
Acknowledgments
We would like to acknowledge funding from the National Science Foundation
Science and Technology Center for Molecular Biotechnology. DRG acknowledges
funding from Kinetek Pharmaceuticals and NIH. Further details of our work
including other references can be found at http://weber.u.washington.edu/~ruedilab.
References
Aebersold R H, Leavitt J, Saavadra R A, Hood L E and Kent S B (1987) Proc Nat! Acad Sci USA
84:6970-6974
Aebersold R H, Teplow DB, Hood L E and Kent S B (1986) J Bioi Chern 261:4229-4238
Affolter M, Watts JD, Krebs DL, Aebersold R (1994) Anal Biochem 15:74-81.
Carr S A, Huddleston M J and Annan R S (1996) Anal Biochem 239:180-192
Carter J R, Franden M A, Aebersold R and McHenry C S (1992) J BacterioI174:7013-7025
Carter J R, Franden M A, Aebersold R and McHenry C S (1993) J Bacteriol 175:5604-5610
Davis M T and Lee T D (1997) J Am Soc Mass Spectrom 9:194-201
Ducret A, van Oostveen I, Eng J K, Yates III J Rand Aebersold R (1998) Protein Sci 7:706-719
Eng J, McCormack A L and Yates III J R (1994) J Amer Soc Mass Spectrom 5:976-989
Figeys D, Ducret A, Yates III J Rand Aebersold R (1996) Nature Biotech 14:1579 - 1583
Figeys D and Aebersold R (1997) Electrophoresis 18:360-368
Figeys D, Ning Y and Aebersold R (1997) Anal Chern 69:3153-3160
Figeys b, Corthals G L, Gallis B, Goodlett D R, Ducret A, Corson M A and Aebersold R (1999) Anal
Chern in press
Fleischmann R D, Adams M D, White 0, Clayton R A, Kirkness E F, Kerlavage A R, Bult C J, Tomb
J -F, Dougherty B A, Merrick J M, McKenney K, Sutton G, FitzHugh W, Fields C, Gocayne J D, Scott
J, Shirley R, L. Liu -I, Glodek A, Kelley J M, Weideman J F, Phillips C A, Spriggs T, Hedblom E, Cotton M D, Utterback T R, Hanna N C, Nguyen D T, Saudek D M, Brandon R C, Fine L D, Fritchman
J L, Fuhrmann J L, Geoghagen N S M, Gnehm C L, McDonald L A, Small K V, Fraser C M, Smith C
° and Venter J C (1995) Science 269:496-512
7
Conclusions
13
While it is apparent that current 2DE technology has limitations for proteome
analysis it is nevertheless extremely useful for large scale protein analysis and for
the analysis of specific proteins in a sample. As noted by an examination of codon
bias values (Gygi et al. 1998) coverage of an entire proteome is currently out of
reach because of the low copy number of most proteins in any given cell. However, those using the technology to ask direct biological questions can be successful (Santos et al. 1998). To provide the best chance of success research questions
involving large scale protein analysis should be addressed using a suitable combination of the available biochemical and immunological tools for isolation and
purification of proteins. As for the future of mass spectrometers in proteomics it
should be possible to identify a protein based on accurate mass measurement of
a single peptide using FT-ICR mass spectrometers. Additionally, the wider use of
MALDI on instruments such as FT-ICR and ion trap mass spectrometers may
facilitate a more judicious use of valuable samples fractionated on-line during
ESI-MS/MS at nanoliter/minute flow rates and then re-analyzed on MALD!
instruments.
8
Acknowledgments
We would like to acknowledge funding from the National Science Foundation
Science and Technology Center for Molecular Biotechnology. DRG acknowledges
funding from Kinetek Pharmaceuticals and NIH. Further details of our work
including other references can be found at http://weber.u.washington.edu/~ruedilab.
References
Aebersold R H, Leavitt J, Saavadra R A, Hood L E and Kent S B (1987) Proc Nat! Acad Sci USA
84:6970-6974
Aebersold R H, Teplow DB, Hood L E and Kent S B (1986) J Bioi Chern 261:4229-4238
Affolter M, Watts JD, Krebs DL, Aebersold R (1994) Anal Biochem 15:74-81.
Carr S A, Huddleston M J and Annan R S (1996) Anal Biochem 239:180-192
Carter J R, Franden M A, Aebersold R and McHenry C S (1992) J BacterioI174:7013-7025
Carter J R, Franden M A, Aebersold R and McHenry C S (1993) J Bacteriol 175:5604-5610
Davis M T and Lee T D (1997) J Am Soc Mass Spectrom 9:194-201
Ducret A, van Oostveen I, Eng J K, Yates III J Rand Aebersold R (1998) Protein Sci 7:706-719
Eng J, McCormack A L and Yates III J R (1994) J Amer Soc Mass Spectrom 5:976-989
Figeys D, Ducret A, Yates III J Rand Aebersold R (1996) Nature Biotech 14:1579 - 1583
Figeys D and Aebersold R (1997) Electrophoresis 18:360-368
Figeys D, Ning Y and Aebersold R (1997) Anal Chern 69:3153-3160
Figeys b, Corthals G L, Gallis B, Goodlett D R, Ducret A, Corson M A and Aebersold R (1999) Anal
Chern in press
Fleischmann R D, Adams M D, White 0, Clayton R A, Kirkness E F, Kerlavage A R, Bult C J, Tomb
J -F, Dougherty B A, Merrick J M, McKenney K, Sutton G, FitzHugh W, Fields C, Gocayne J D, Scott
J, Shirley R, L. Liu -I, Glodek A, Kelley J M, Weideman J F, Phillips C A, Spriggs T, Hedblom E, Cotton M D, Utterback T R, Hanna N C, Nguyen D T, Saudek D M, Brandon R C, Fine L D, Fritchman
J L, Fuhrmann J L, Geoghagen N S M, Gnehm C L, McDonald L A, Small K V, Fraser C M, Smith C
° and Venter J C (1995) Science 269:496-512