A rapid method for analyzing recombinant protein inclusion bodies by mass spectrometry.
The identification and characterization of a protein overexpressed in insoluble inclusion bodies in Escherichia coli are the first crucial and time-limiting steps in recombinant protein expression. Here, a straightforward approach to the analysis of recombinant proteins in inclusion bodies is presented. Inclusion bodies were dissolved in 8M urea and analyzed by matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry without prior desalting. Mass determination was achieved by direct spotting of the samples onto the MALDI target and serial dilution in the matrix. The masses of four different proteins, expressed in inclusion bodies, were determined with a mass accuracy better than 0.1%. Furthermore, protein modifications, such as N-terminal processing of single amino acids or artificial cyanylation caused by incubation of the inclusion bodies with urea at elevated temperatures, could be detected. Similarly, tryptic digests were directly analyzed in 2M urea to obtain peptide mass fingerprints for identification and more detailed information on the primary protein structure and secondary modifications. Due to the presence of ammonia in the urea-containing buffers, no Na(+) adducts were observed in the peptide mass fingerprint analysis. Taken together, the rapid and robust procedures presented here greatly facilitate the analysis of recombinant proteins.
Grimm
Rudolf
R
Hexal Biotech ForschungsGmbH, Keltenring 1+3, 82041 Oberhaching, Germany.
Huber
Robert
R
Neumeier
Thomas
T
Seidl
Andreas
A
Haslbeck
Martin
M
Seibert
Fabian S
FS
D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins