Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.
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.
Wilm
M
M
Protein & Peptide Group, EMBL, Heidelberg, Germany.
Shevchenko
A
A
Houthaeve
T
T
Breit
S
S
Schweigerer
L
L
Fotsis
T
T
Mann
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D006131:Growth Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry; D017421:Sequence Analysis / Q000379:methods*; D012710:Serum Albumin, Bovine; D014407:Tumor Cells, Cultured
D006131:Growth Inhibitors; D011506:Proteins; D016601:RNA-Binding Proteins; D012710:Serum Albumin, Bovine; D004247:DNA