TY - JOUR

T1 - Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry

AU - Wilm, M

AU - Shevchenko, A

AU - Houthaeve, T

AU - Breit, S

AU - Schweigerer, L

AU - Fotsis, T

AU - Mann, M

PY - 1996/2/1

Y1 - 1996/2/1

N2 - Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.

AB - Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.

KW - Amino Acid Sequence

KW - Base Sequence

KW - Cloning, Molecular

KW - DNA

KW - Electrophoresis, Polyacrylamide Gel

KW - Endothelium, Vascular/cytology

KW - Growth Inhibitors/chemistry

KW - Humans

KW - Mass Spectrometry

KW - Molecular Sequence Data

KW - Proteins/chemistry

KW - RNA-Binding Proteins/chemistry

KW - Sequence Analysis/methods

KW - Serum Albumin, Bovine

KW - Tumor Cells, Cultured

U2 - 10.1038/379466a0

DO - 10.1038/379466a0

M3 - Journal article

C2 - 8559255

VL - 379

SP - 466

EP - 469

JO - Nature

JF - Nature

SN - 0028-0836

IS - 6564

ER -