Identification and Characterization of Proteins based on Mass Spectrometric Mapping of Tryptic Peptides and On-line Internet Database Searches
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Series / Report no.
Open Access
Type
Report
Language
en
Date
1997-11-30
Research Projects
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Journal Issue
Title
Identification and Characterization of Proteins based on Mass Spectrometric Mapping of Tryptic Peptides and On-line Internet Database Searches
Translated Title
Identificatie en karakterisering van eiwitten gebaseerd op massa spectrometrische analyse van peptiden na trypsine digestie en het on-line zoeken op internet in bestanden
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Abstract
Er is een methode beschreven voor de identificatie en karakterisering van eiwitten, welke geisoleerd zijn met behulp van een- of tweedimensionale polyacrylamide gel elektroforese. De methode is gebaseerd op moderne technieken met betrekking tot concentrering en zuivering van eiwitten (elektro-elutie van eiwitten naar een secundaire gel en het reversed phase micro capillaire kolom schakel HPLC principe) gevolgd door massaspectrometrische analyse met behulp van nano-elektrospray ionisatie en de quadrupool ion trap. De beschreven studies zijn uitgevoerd met Bovine Serum Albumine (BSA) als modeleiwit. Met de beschreven elektro-elutie methode kan een concentreringsfactor van het eiwit van tenminste 50 verkregen worden. Dit kan nog verhoogd worden door eiwitspots uit meerdere primaire gels te combineren op een secundaire gel. Op deze wijze kunnen kleine hoeveelheden aan eiwit (lage femtomol gebied) geanalyseerd worden. Tevens wordt gebruik gemaakt van het internet om bestanden van bekende eiwitten on-line te raadplegen, en de uit de MS(n) experimenten bepaalde (gedeeltelijke) aminozuursequentie van de peptiden te correleren aan deze bekende eiwitten. Ingegaan wordt op enige praktische aspecten van de methode, met betrekking tot de matrix van de eiwitmonsters en eventuele modificaties van de peptiden na trypsine digestie.<br>
A method described for the identification and characterization of proteins isolated on primary one- or two-dimensional polyacrylamide gel electrophoresis has been adapted to recently developed concentration/desalinization techniques (electrotransferation of proteins to a secondary gel and reversed-phase micro-capillary column switching trapping system), and mass spectrometric techniques like nano-electrospray ionization in combination with quadrupole ion-trap mass spectrometry. Model studies were performed with Bovine Serum Albumin. It was found using this method that concentration factors of at least 50-fold can be easily obtained by electro-transfering the isolated proteins on a secondary gel. Low femtomole amounts can be sequenced when spots from multiple primary gels are combined on a single secondary gel. On-line Internet database searches are used to identify the protein by using (partial) sequence information obtained from MS(n) experiments of the peptides after tryptic proteolysis. Different aspects of the applicability of the method, like sample matrices and chemical modifications of the tryptic peptides to the MS analysis, were also considered.<br>
A method described for the identification and characterization of proteins isolated on primary one- or two-dimensional polyacrylamide gel electrophoresis has been adapted to recently developed concentration/desalinization techniques (electrotransferation of proteins to a secondary gel and reversed-phase micro-capillary column switching trapping system), and mass spectrometric techniques like nano-electrospray ionization in combination with quadrupole ion-trap mass spectrometry. Model studies were performed with Bovine Serum Albumin. It was found using this method that concentration factors of at least 50-fold can be easily obtained by electro-transfering the isolated proteins on a secondary gel. Low femtomole amounts can be sequenced when spots from multiple primary gels are combined on a single secondary gel. On-line Internet database searches are used to identify the protein by using (partial) sequence information obtained from MS(n) experiments of the peptides after tryptic proteolysis. Different aspects of the applicability of the method, like sample matrices and chemical modifications of the tryptic peptides to the MS analysis, were also considered.<br>
Description
Publisher
Rijksinstituut voor Volksgezondheid en Milieu RIVM
Sponsors
RIVM