86
A.-CH. BERGMAN et al.
the level of the background. The sample was analyzed for eleven more cycles and
the sequence could be clearly assigned. This was also true for residue 12, valine,
that was recovered in only lO femtomole (Table 6.1). A database search using the
BLAST algorithm identified this polypeptide as the 78 kDa glucose regulated protein (GRP) having SwissProt accession number PIlO2l. This protein belongs to
the heat shock protein 70 family and is located in the lumen of the endoplasmic
reticulum. GRP probably plays a role in the assembly of protein complexes inside
the endoplasmic reticulum. Interestingly, an N-terminally truncated form of GRP
was found by Rasmussen et al. (1997) when this protein was isolated via 2-D gel
electrophoresis applied to a human breast carcinoma cell line. The first three glutamic acid residues were lacking and the sequence started at residue 4, aspartic
acid (cf. Table 6.1). The results show that the sub-picomole sequencer capability
makes electroblotting to PVDF after 2-D gel separation an attractive alternative
for rapid identification via direct sequence analysis of many cellular proteins isolated in amounts down to about 1 picomole.
3
Conclusion
Mass spectrometry and high sensitivity Edman degradation are both important
analytical tools for characterization of gel separated proteins (Fig. 6.1). Mass
mapping for tentative identification and CID for verification is efficient when the
protein is known and in the database. For cloning and when longer sequence
stretches are required, high sensitivity Edman degradation is a strong alternative
provided amounts are not too low, minimally 3-5 picomole in the gel. However,
for intact proteins electroblotted to PVDF, the amount can be substantially
smaller (0.5-1 picomole).
4
Acknowledgements
This work was supported by grants from the Swedish Medical Research Council
(project 03X-10832), the Swedish Cancer Society (project 1806), the European
Commission (BI04-CT97 - 2123) and the Emil and Wera Cornell Foundation.
References
Altschul SF, Gish W (1996) Local alignment statistics. Meth Enzymol 266: 460-480
Bergman A-C, Linder C, Sakaguchi K, Sten-Linder M, Alaiya AA, Franzen B, Shoshan MC, Bergman
T, Wiman B, Auer G, Appella E, Jornvall H, Linder S (1997) Increased expression of a-enolase in cjun transformed rat fibroblasts without increased activation of plasminogen. FEBS Lett 417: 17-20
Bergman A-C, Oppermann M, Jornvall H, Bergman T (1998) Mass mapping and sensitive Edman
degradation of gel separated proteins. J Prot Chern 17: 535-536
Bergman A-C, Alaiya AA, Wendler W, Binetruy B, Shoshan M, Sakaguchi K, Bergman T, Kronenwett
U, Auer G, Appella E, Jornvall H, Linder S (1999a) Protein kinase dependent overexpression of the
nuclear protein Pirin in c-JUN and RAS transformed fibroblasts. Cellular and Molecular Life Sciences, in press
Bergman A-C, Benjamin T, Alaiya A, Waltham M, Sakaguchi K, Franzen B, Bergman T, Linder S, Auer
G, Appella E, Wirth pJ, Jornvali H (1999b) Identification of gel-separated tumor marker proteins by
mass spectrometry. Electrophoresis, submitted
A.-CH. BERGMAN et al.
the level of the background. The sample was analyzed for eleven more cycles and
the sequence could be clearly assigned. This was also true for residue 12, valine,
that was recovered in only lO femtomole (Table 6.1). A database search using the
BLAST algorithm identified this polypeptide as the 78 kDa glucose regulated protein (GRP) having SwissProt accession number PIlO2l. This protein belongs to
the heat shock protein 70 family and is located in the lumen of the endoplasmic
reticulum. GRP probably plays a role in the assembly of protein complexes inside
the endoplasmic reticulum. Interestingly, an N-terminally truncated form of GRP
was found by Rasmussen et al. (1997) when this protein was isolated via 2-D gel
electrophoresis applied to a human breast carcinoma cell line. The first three glutamic acid residues were lacking and the sequence started at residue 4, aspartic
acid (cf. Table 6.1). The results show that the sub-picomole sequencer capability
makes electroblotting to PVDF after 2-D gel separation an attractive alternative
for rapid identification via direct sequence analysis of many cellular proteins isolated in amounts down to about 1 picomole.
3
Conclusion
Mass spectrometry and high sensitivity Edman degradation are both important
analytical tools for characterization of gel separated proteins (Fig. 6.1). Mass
mapping for tentative identification and CID for verification is efficient when the
protein is known and in the database. For cloning and when longer sequence
stretches are required, high sensitivity Edman degradation is a strong alternative
provided amounts are not too low, minimally 3-5 picomole in the gel. However,
for intact proteins electroblotted to PVDF, the amount can be substantially
smaller (0.5-1 picomole).
4
Acknowledgements
This work was supported by grants from the Swedish Medical Research Council
(project 03X-10832), the Swedish Cancer Society (project 1806), the European
Commission (BI04-CT97 - 2123) and the Emil and Wera Cornell Foundation.
References
Altschul SF, Gish W (1996) Local alignment statistics. Meth Enzymol 266: 460-480
Bergman A-C, Linder C, Sakaguchi K, Sten-Linder M, Alaiya AA, Franzen B, Shoshan MC, Bergman
T, Wiman B, Auer G, Appella E, Jornvall H, Linder S (1997) Increased expression of a-enolase in cjun transformed rat fibroblasts without increased activation of plasminogen. FEBS Lett 417: 17-20
Bergman A-C, Oppermann M, Jornvall H, Bergman T (1998) Mass mapping and sensitive Edman
degradation of gel separated proteins. J Prot Chern 17: 535-536
Bergman A-C, Alaiya AA, Wendler W, Binetruy B, Shoshan M, Sakaguchi K, Bergman T, Kronenwett
U, Auer G, Appella E, Jornvall H, Linder S (1999a) Protein kinase dependent overexpression of the
nuclear protein Pirin in c-JUN and RAS transformed fibroblasts. Cellular and Molecular Life Sciences, in press
Bergman A-C, Benjamin T, Alaiya A, Waltham M, Sakaguchi K, Franzen B, Bergman T, Linder S, Auer
G, Appella E, Wirth pJ, Jornvali H (1999b) Identification of gel-separated tumor marker proteins by
mass spectrometry. Electrophoresis, submitted